Cytotoxic agents containing novel potent taxanes and their therapeutic use

ABSTRACT

Included within the scope of the present invention are potent taxanes containing a linking group. Also included is a cytotoxic agent comprising one or more taxanes linked to a cell binding agent. A therapeutic composition for inducing cell death in selected cell populations comprising: (A) a cytotoxic amount of one or more taxanes covalently bonded to a cell binding agent through a linking group, and (B) a pharmaceutically acceptable carrier, diluent or excipient is also included. A method for inducing cell death in selected cell populations comprising contacting target cells or tissue containing target cells with an effective amount of a cytotoxic agent comprising one or more taxanes linked to a cell binding agent is included as well.

[0001] This is a continuation-in-part of U.S. Ser. No. 10/210,112, filedAug. 2, 2002, which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to novel cytotoxic agents and theirtherapeutic use. More specifically, the invention relates to noveltaxanes, novel cytotoxic agents comprising the novel taxanes and theirtherapeutic use. These novel cytotoxic agents have therapeutic use inthat taxanes are delivered to a selected cell population in a targetedfashion by chemically linking the taxanes to a cell-binding agent thatis able to target the selected cell population.

BACKGROUND OF THE INVENTION

[0003] The specificity of cytotoxic agents can be greatly improved bytargeted delivery through linkage of the cytotoxic agents tocell-binding agents.

[0004] Many reports have appeared on the attempted specific targeting oftumor cells with monoclonal antibody-drug conjugates (Sela et al, inImmunoconjugates 189-216 (C. Vogel, ed. 1987); Ghose et al, in TargetedDrugs 1-22 (E. Goldberg, ed. 1983); Diener et al, in Antibody mediateddelivery systems 1-23 (J. Rodwell, ed. 1988); Pietersz et al, inAntibody mediated delivery systems 25-53 (J. Rodwell, ed. 1988); Bumolet al, in Antibody mediated delivery systems 55-79 (J. Rodwell, ed.1988). All references and patents cited herein are incorporated byreference.

[0005] Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin,vincristine, vinblastine, melphalan, mitomycin C, and chlorambucil havebeen conjugated to a variety of murine monoclonal antibodies. In somecases, the drug molecules were linked to the antibody molecules throughan intermediary carrier molecule such as serum albumin (Garnett et al,Cancer Res. 46: -2412 (1986); Ohkawa et al, Cancer Immunol. Immunother.23: 86 (1986); Endo et al, Cancer Res. 47: 1076-1080 (1980)), dextran(Hurwitz et al, Appl. Biochem. 2: 25-35 (1980); Manabi et al, Biochem.Pharmacol. 34: 289-291 (1985); Dillman et al, Cancer Res. 46: 4886-4891(1986); Shoval et al, Proc. Natl. Acad. Sci. 85: 8276-8280 (1988)), orpolyglutamic acid (Tsukada et al, J. Natl. Canc. Inst. 73: 721-729(1984); Kato et al, J. Med. Chem. 27: 1602-1607 (1984); Tsukada et al,Br. J. Cancer 52: 111-116 (1985)).

[0006] A wide array of linker technologies has been employed for thepreparation of such immunoconjugates and both cleavable andnon-cleavable linkers have been investigated. In most cases, the fullcytotoxic potential of the drugs could only be observed, however, if thedrug molecules could be released from the conjugates in unmodified format the target site.

[0007] One of the cleavable linkers that has been employed for thepreparation of antibody-drug conjugates is an acid-labile linker basedon cis-aconitic acid that takes advantage of the acidic environment ofdifferent intracellular compartments such as the endosomes encounteredduring receptor mediated endocytosis and the lysosomes. Shen and Ryserintroduced this method for the preparation of conjugates of daunorubicinwith macromolecular carriers (Biochem. Biophys. Res. Commun. 102:1048-1054 (1981)). Yang and Reisfeld used the same technique toconjugate daunorubicin to an anti-melanoma antibody (J. Natl. Canc.Inst. 80: 1154-1159 (1988)). Dillman et al. also used an acid-labilelinker in a similar fashion to prepare conjugates of daunorubicin withan anti-T cell antibody (Cancer Res. 48: 6097-6102 (1988)).

[0008] An alternative approach, explored by Trouet et al, involvedlinking daunorubicin to an antibody via a peptide spacer arm (Proc.Natl. Acad. Sci. 79: 626-629 (1982)). This was done under the premisethat free drug could be released from such a conjugate by the action oflysosomal peptidases. In vitro cytotoxicity tests, however, haverevealed that antibody-drug conjugates rarely achieved the samecytotoxic potency as the free unconjugated drugs. This suggested thatmechanisms by which drug molecules are released from the antibodies arevery inefficient.

[0009] In the area of immunotoxins, conjugates formed via disulfidebridges between monoclonal antibodies and catalytically active proteintoxins were shown to be more cytotoxic than conjugates containing otherlinkers. See, Lambert et al, J. Biol. Chem. 260: 12035-12041 (1985);Lambert et al, in Immunotoxins 175-209 (A. Frankel, ed. 1988); Ghetie etal, Cancer Res. 48: 2610-2617 (1988). This was attributed to the highintracellular concentration of glutathione contributing to the efficientcleavage of the disulfide bond between an antibody molecule and a toxin.Despite this, there are only a few reported examples of the use ofdisulfide bridges for the preparation of conjugates between drugs andmacromolecules. (Shen et al, J. Biol. Chem. 260: 10905-10908 (1985))described the conversion of methotrexate into a mercaptoethylamidederivative followed by conjugation with poly-D-lysine via a disulfidebond. Another report described the preparation of a conjugate of thetrisulfide containing toxic drug calicheamycin with an antibody (Hinmanet al, Cancer Res. 53: 3336-3342 (1993)).

[0010] One reason for the lack of disulfide linked antibody-drugconjugates is the unavailability of cytotoxic drugs possessing a sulfuratom containing moiety that can be used readily to link the drug to anantibody via a disulfide bridge. Furthermore, chemical modification ofexisting drugs is difficult without diminishing their cytotoxicpotential.

[0011] Another major drawback with existing antibody-drug conjugates istheir inability to deliver a sufficient concentration of drug to thetarget site because of the limited number of targeted antigens and therelatively moderate cytotoxicity of cancerostatic drugs likemethotrexate, daunorubicin, and vincristine. In order to achievesignificant cytotoxicity, linkage of a large number of drug molecules,either directly to the antibody or through a polymeric carrier molecule,becomes necessary. However, such heavily modified antibodies oftendisplay impaired binding to the target antigen and fast in vivoclearance from the blood stream.

[0012] In spite of the above-described difficulties, useful cytotoxicagents comprising cell-binding moieties and the group of cytotoxic drugsknown as maytansinoids have been reported (U.S. Pat. Nos. 5,208,020,5,416,064, and R. V. J. Chari, Advanced Drug Delivery Reviews 31: 89-104(1998)). Similarly, useful cytotoxic agents comprising cell-bindingmoieties and analogues and derivatives of the potent antitumorantibiotic CC-1065 have also been reported (U.S. Pat. Nos. 5,475,092 and5,585,499).

[0013] It has also been shown that the linkage of highly cytotoxic drugsto antibodies using a cleavable link, such as a disulfide bond, ensuresthe release of fully active drug inside cells, and such conjugates arecytotoxic in an antigen specific manner (R. V. J. Chari et al, CancerRes. 52: 127-131 (1992); U.S. Pat. Nos. 5,475,092; and 5,416,064).

[0014] Taxanes are a family of compounds that includes paclitaxel(Taxol), a cytotoxic natural product, and docetaxel (Taxotere), asemi-synthetic derivative (see FIGS. 1 and 4), two compounds that arewidely used in the treatment of cancer, E. Baloglu and D. G. I.Kingston, J. Nat. Prod. 62: 1448-1472 (1999). Taxanes are mitoticspindle poisons that inhibit the depolymerization of tubulin, resultingin cell death. While docetaxel and paclitaxel are useful agents in thetreatment of cancer, their antitumor activity is limited because oftheir non-specific toxicity towards normal cells. Further, compoundslike paclitaxel and docetaxel themselves are not sufficiently potent tobe used in conjugates of cell-binding agents.

[0015] Recently, a few new docetaxel analogs with greater potency thaneither docetaxel or paclitaxel have been described (I. Ojima et al, J.Med. Chem., 39: 3889-3896 (1996)). However, these compounds lack asuitable functionality that allows linkage via a cleavable bond tocell-binding agents (FIG. 1).

[0016] The synthesis of novel taxanes that retain high cytotoxicity andthat can be effectively linked to cell-binding agents has been describedrecently (U.S. Pat. Nos. 6,340,701, 6,372,738 and 6,436,931, and FIGS. 2and 4). In these disclosures, taxanes were modified with chemicalmoieties, ones containing thiol or disulfide groups in particular, towhich appropriate cell-binding agents could be linked. As a result,these novel taxanes preserved, and in some cases even enhanced, thecytotoxic potency of known taxanes.

[0017] In the taxanes described in the aforementioned patents, thelinking group was introduced at the C-10, C-7 or the C-2′ position ofthe taxane.

[0018] In the cases where the linking group was at C-7, the C-10position did not have a free hydroxyl substituent but was rather anester, ether or carbamate substituent. It has been previously shown (I.Ojima et al, J. Med Chem., 39: 3889-3896 (1996)) that the presence of anester or carbamate substituent at C-10 produced taxoids of high potency.However, there have been no studies on the potency of taxanes bearing afree hydroxyl group at C-10 and a linking group at C-7.

[0019] As described herein, the potency of taxanes bearing a freehydroxyl group at C-10 and a linking group at C-7 was found to meet orexceed the potency of taxanes bearing an ester, ether or carbamatesubstituent at C-10 and a linking group at C-7. Thus, in a first aspect,the present invention provides these novel taxanes bearing a freehydroxyl group at C-10 and a linking group at C-7 and having potentcytotoxic activity.

[0020] Further, in the taxanes described in the aforementioned patents,the linking group was introduced at the C-10, C-7 or the C-2′ positionof the taxane. In all taxanes, the substituents at C-3′N and C-3′, werenamed -NHCOR₄ and R₃, respectively. Furthermore, the substituent atC-3′N, —NHCOR₄, was either a benzamido group (R₄=phenyl), like inpaclitaxel, or a tert-butyloxycarbonylamino moiety (—NH-t-BOC,R₄=t-BOC), like in docetaxel. Based on published data, it was assumedthat altering these substituents would cause a loss in potency. Thesubstituent at C-3′ (R₃) was either aryl or a linear, branched or cyclicalkyl group having 1 to 10 carbon atoms. Since, based on published data,it was thought that the substituents at C-3′N and C-3′ could not bealtered without a loss in drug activity, the linking group was alwaysintroduced at a different position of the taxane, namely at C-7, C-10 orC-2′. Also, the inability to change the substituents at C-3′ or C-3′Ngreatly limited the variety of disulfide-containing taxanes that couldbe synthesized.

[0021] In a second aspect, the present invention is also based on theunexpected finding that the substituents at both C-3′ and C-3′N do nothave to be limited to that present in the known taxanes. As describedherein, the potency of taxanes bearing a variety of differentsubstituents at C-3′N meets or exceeds the potency of taxanes bearing abenzamido or —NH-t-BOC substituent at this position. The new substituentat C-3′ or C-3′N can also contain a linking group that allows forlinkage to cell-binding agents. The present invention discloses thesenovel highly potent taxanes bearing a variety of different substituentsat C-3′ and C-3′N. The linking group can now be incorporated at any oneof the five positions: C-3′, C-3′N, C-10, C-7 or C-2′.

SUMMARY OF THE INVENTION

[0022] In one embodiment of the present invention, novel taxanes thatare highly cytotoxic and that can still be effectively used in thetreatment of many diseases are disclosed.

[0023] In a second embodiment of the present invention, novel taxanesthat bear a free hydroxyl group at the C-10 position and a linking groupat C-7, and that still maintain high potency, are disclosed.

[0024] In a third embodiment of the present invention, novel taxanesthat bear a variety of different substituents at C-3′N or C-3′, and alinking group at C-7, C-10, C-2′ or C-3′N or C-3′ and that stillmaintain high potency, are disclosed.

[0025] In a fourth embodiment of the present invention, a cytotoxicagent comprising one or more novel taxanes covalently bonded to acell-binding agent through a linking group is disclosed.

[0026] In a fifth embodiment of the present invention, a therapeuticcomposition comprising:

[0027] (a) a therapeutically effective amount of one or more noveltaxanes linked to a cell-binding agent, and

[0028] (b) a pharmaceutically acceptable carrier, diluent, or excipient,is disclosed.

[0029] In a sixth embodiment of the present invention, a method ofinducing cell death in selected cell populations comprising contactingtarget cells or tissue containing target cells, with an effective amountof a cytotoxic agent comprising one or more novel taxanes linked to acell-binding agent is disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 is a chemical formula that represents structures of varioustaxanes, including some of the more potent taxanes described by Ojima etal., supra.

[0031]FIG. 2 is a chemical formula that represents structures of some ofthe disulfide-containing taxanes according to the present invention thatbear a free hydroxyl group at the C-10 position and a linking group atC-7.

[0032]FIG. 3 shows the structure of three taxanes. Taxane 1 bears anester group at C-10 and a linking group at C-7. Both taxane 2′ andtaxane 3′ bear a free hydroxy group at C-10 and a linking group at C-7.

[0033]FIG. 4 is a chemical formula that represents structures of varioustaxanes, including some of the more potent taxanes described in U.S.Pat. Nos. 6,340,701, 6,372,738 and 6,436,931.

[0034]FIG. 5 is a chemical formula that represents structures of some ofthe new taxanes according to the present invention that have asubstituent at R₃ and/or R₄ not previously described.

[0035]FIG. 6 is a chemical formula that represents structures of some ofthe new disulfide-containing taxanes according to the present inventionthat have a substituent at R₃ and/or R₄ not previously described.

[0036]FIG. 7 shows the structure of 10-deacetylbaccatin III, which isthe starting material for preparing taxanes.

[0037]FIG. 8 shows the synthetic steps in the production of taxane 2′.

[0038]FIG. 9 shows the synthetic steps in the production of taxane 3′.

[0039]FIG. 10 shows a comparison of the in vitro potency of taxanes 1and 2′ towards A431 cells.

[0040]FIG. 11 shows the in vitro cytotoxicity of taxane 3′ toward A549and MCF-7 cells.

[0041]FIG. 12 shows the anti-tumor effect of anti-EGF receptorantibody-taxane conjugate on human squamous cancer (A431) xenografts inSCID mice.

[0042]FIG. 13 shows the body weight change of the SCID mice used in theexperiment described in Example 8.

[0043]FIG. 14 shows the results of a cytotoxicity determination for theanti-EGF receptor-taxane conjugate on the target antigen-positive cellline A431 and for the N901-taxane conjugate for which the A431 cell linedoes not express the target antigen.

[0044]FIG. 15 shows the cytotoxic potency and selectivity of theTA1-taxane conjugate in the target antigen-positive cell line SK-BR-3and the non-target antigen-negative cell line A431.

[0045]FIGS. 16a, 16 b and 16 c show the synthetic steps in theproduction of new taxanes according to the second aspect of the presentinvention.

[0046]FIGS. 17a and 17 b show the synthetic steps in the production ofnew disulfide-containing taxanes according to the second aspect of thepresent invention.

[0047]FIG. 18 shows the in vitro cytotoxicity of new taxanes accordingto the second aspect of the present invention

[0048]FIGS. 19a and 19 b show the in vitro cytotoxicity ofdisulfide-containing taxanes according to the second aspect of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

[0049] The present invention describes novel taxanes that retain highcytotoxicity and that can be effectively linked to cell binding agents.It has previously been shown that taxanes possessing a protectedhydroxyl group at C-10 are highly potent (U.S. Pat. Nos. 6,340,701,6,372,738 and 6,436,931. The first aspect of the present invention isbased on the unexpected finding that the C-10 position does not have tobe protected to attain high potency. Taxanes bearing a free hydroxygroup at C-10 still maintain high potency as long as there is aprotected hydroxy group at C-7, such as a linking group.

[0050] The present invention further describes the synthesis and invitro evaluation of taxanes bearing a free hydroxyl group at C-10 and alinking group at C-7.

[0051] Also, it has previously been shown that taxanes possessing abenzamido or a tert-butyloxycarbonylamino (—NH-t-BOC) substituent atC-3′N along with another substituent which is an aryl or a linearbranched or cyclic alkyl group are highly potent. The second aspect ofthe invention is based on the unexpected finding that the C-3′N positiondoes not have to possess a benzamido or a —NH-t-BOC group to attain highpotency. A number of different amide or carbamate substituents bearingalkyl, alkenyl or heterocyclic side chains can be used without any lossin potency. The linking group can be introduced on the side chains atC-3′, C-3′N, or on the C-10, C-7 or C-2′ positions.

[0052] The precursor to the synthesis of taxanes is the naturallyoccurring compound 10-deacetylbaccatin III (10-DAB) (FIG. 7). A largevariety of taxanes bearing a linking group can be prepared. Further,this compound has a free hydroxyl group at the C-10 position. Therefore,the number of synthetic steps needed for the production of a cytotoxictaxane according to the first aspect of the invention can be decreasedbecause the hydroxyl group does not have to be converted into an ester,ether or carbamate. The yield of taxanes bearing a linking group canalso be increased.

[0053] The present invention further describes the synthesis and invitro evaluation of representative taxanes bearing new substituents atC-3′ or C-3′N, with or without a linking group at C-7, C-10, C-2′ or atC-3′, C-3′N.

[0054] The art reveals that it is extremely difficult to modify existingdrugs without diminishing their cytotoxic potential. The disclosedinvention overcomes this problem by modifying the disclosed taxanes withchemical moieties, including ones containing thiol or disulfide groups,to which appropriate cell-binding agents can be linked. As a result, thedisclosed novel taxanes preserve, and in some cases could even enhance,the cytotoxic potency of known taxanes. The cell-binding agent-taxanecomplexes permit the full measure of the cytotoxic action of the taxanesto be applied in a targeted fashion against unwanted cells only,therefore, avoiding side effects due to damage to non-targeted healthycells. This invention permits the taxanes to be target site-directed,which had been impossible previously. Thus, the invention providesuseful agents for the elimination of diseased or abnormal cells that areto be killed or lysed, such as tumor cells (particularly solid tumorcells), virus infected cells, microorganism infected cells, parasiteinfected cells, autoimmune cells (cells that produce autoantibodies orcells that regulate the production of autoantibodies), activated cells(those involved in graft rejection or graft vs. host disease), or anyother type of diseased or abnormal cells, while exhibiting a minimum ofside effects.

[0055] The cytotoxic agent according to the present invention comprisesone or more taxanes linked to a cell-binding agent via a linking group.The linking group is part of a chemical moiety that is covalently boundto a taxane through conventional methods. In a preferred embodiment, thecell-binding agent can be covalently bound to the taxane via a disulfideor a thioether linkage.

[0056] In the following description of embodiments (1) to (9), thefollowing apply:

[0057] The term “alkyl” means linear, branched or cyclic, unlessotherwise specified.

[0058] Examples of linear alkyls include methyl, ethyl, propyl, butyl,pentyl and hexyl.

[0059] Examples of branched alkyls include isopropyl, isobutyl,sec.-butyl, tert.-butyl, isopentyl and 2-ethyl-propyl.

[0060] Examples of cyclic alkyls include cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

[0061] Examples of alkenyls and cycloalkenyls include dimethylacryloyl,isobutenyl, hexenyl, cyclopentenyl, and cyclohexenyl.

[0062] Examples of simple aryls include phenyl and naphthyl.

[0063] Examples of substituted aryls include aryls such as thosedescribed above substituted with alkyl groups, with halogens, such asCl, Br, F, nitro groups, amino groups, sulfonic acid groups, carboxylicacid groups, hydroxy groups and alkoxy groups.

[0064] Heterocyclics are compounds wherein the heteroatoms are selectedfrom O, N, and S, and include morpholino, piperidino, piperazino,N-methylpiperazino, pyrrollyl, pyridyl, furyl, imidazolyl, oxazolyl,thiazolyl and thiopheneyl, indolyl, benzofuranyl, benzothiopheneyl.

[0065] Examples of carbamates are those formed from alkyl, alkenyl,cycloalkyl, cycloalkenyl, or aryl moieties, such as, methyl, ethyl,crotonyl, cyclohexyl, cyclohexenyl, phenyl, or from nitrogen-containingheterocycles, such as morpholino, piperidino, piperazino, N-methylpiperazino.

[0066] Examples of aryl esters, ethers, and carbamates include phenyl,and napthyl, ethers, esters and carbamates.

[0067] Examples of linear, branched or cyclic alkyl or alkenyl estersinclude methyl, ethyl, isopropyl, allyl, crotonyl, cyclohexyl,cyclohexenyl esters.

[0068] Examples of linear, branched or cyclic alkyl or alkenyl ethersinclude methyl, ethyl, isopropyl, allyl, crotonyl, and cyclohexylethers.

[0069] The taxanes useful in the present invention have the formula (I)shown below:

[0070] These novel taxanes can be divided into nine embodiments,designated (1) to (9). Examples of the embodiments (1) to (4) are shownin FIG. 2. Examples of embodiments (5) to (9) are shown in FIG. 6.

[0071] Embodiments (1)-(4)

[0072] In embodiments (1) to (4), R₁ is H, an electron withdrawinggroup, such as F, NO₂, CN, Cl, CHF₂, and CF₃ or an electron donatinggroup such as —OCH₃, —OCH₂CH₃, —NR₇R₈, —OR₉. R₁′ and R₁″ are the same ordifferent and are H, an electron withdrawing group, such as F, NO₂, CN,Cl, CHF₂, and CF₃ or an electron donating group such as —OCH₃, —OCH₂CH₃,—NR₇R₈, —OR₉.

[0073] R₇ and R₈ are the same or different and are linear, branched, orcyclic alkyl groups having from 1 to 10 carbon atoms or simple orsubstituted aryl. Preferably the number of carbon atoms for R₇ and R₈ is1 to 4. Also, preferably R₇ and R₈ are the same. Examples of preferred—NR₇R₈ groups include dimethyl amino, diethyl amino, di-isopropyl aminoand dibutyl amino, where the butyl moiety is any of primary, secondary,tertiary or isobutyl.

[0074] R₉ is linear, branched or cyclic alkyl having 1 to 10 carbonatoms. R₁ is preferably —OCH₃, Cl, F, NO₂, and CF₃.

[0075] More preferably, R₁ is —OCH₃ and is in the meta position, and oneof R₁′ and R₁″ is —OCH₃ and the other is H.

[0076] In embodiments (1), (2) and (4), R₂ is H, a heterocyclic, or arylether, ester or carbamate or a linear, branched, or cyclic alkyl oralkenyl ester or ether having from 1 to 10 carbon atoms, or a carbamateof the formula COX, wherein X is a nitrogen-containing heterocycle, suchas piperidino, morpholino, piperazino, N-methyl-piperazino, or acarbamate of the formula —CONR₁₀R₁₁, wherein R₁₀ and R₁₁ are the same ordifferent and are H, linear, branched, or cyclic alkyl having 1 to 10carbon atoms or simple or substituted aryl.

[0077] Preferred examples of aryl ethers, esters, and carbamates includephenyl and naphthyl.

[0078] Preferred examples of alkyl and alkenyl esters include —COCH₃,—COCH₂CH₃, crotonyl and dimethylacryloyl. Preferred examples of alkyland alkenyl ethers include methyl, ethyl, allyl, propyl, crotonyl, anddimethyacryloyl . Preferred examples of carbamates include —CONHCH₂CH₃,—CONHCH₂CH₂CH₃, —CO-morpholino, —CO-piperazino, —CO-piperidino, or—CO-N-methylpiperazino. Preferably, R₂ is H.

[0079] In embodiment (3), R₂ is the linking group.

[0080] In embodiments (1), (3) and (4), R₃ is alkyl or alkenyl, havingfrom 1 to 10 carbon atoms, cycloalkyl, cycloalkenyl having from 3 to 10carbon atoms, aryl, or heterocycle.

[0081] Preferably, R₃ is crotonyl, dimethylacryloyl, isobutenyl,hexenyl, cyclopentenyl, cyclohexenyl, furyl, pyrollyl, thiopheneyl,thiazolyl, imidazolyl, pyridyl, morpholino, piperidino, piperazino,oxazolyl, indolyl, benzofuranyl or benzothiopheneyl.

[0082] More preferably, R₃ is t-BOC, iso-butenyl, crotonyl,dimethyacryloyl, thiophenyl, thiazolyl or furyl.

[0083] In embodiment (2), R₃ is —CH═C(CH₃)₂.

[0084] In embodiments, (1) to (4), R₄ is alkyl or alkenyl, having from 1to 10 carbon atoms, cycloalkyl, cycloalkenyl having from 3 to 10 carbonatoms, aryl, heterocycle, —OC(CH₃)₃ or a carbamate formed from any ofsaid alkyl, alkenyl, cycloalkyl, or cycloalkenyl, aryl, or anitrogen-containing heterocycle.

[0085] Preferably, R₄ is crotonyl, dimethylacryloyl, isobutenyl,hexenyl, cyclopentenyl, cyclohexenyl, furyl, pyrollyl, thiopheneyl,thiazolyl, imidazolyl, pyridyl, morpholino, piperidino, piperazino,oxazolyl, indolyl, benzofuranyl or benzothiopheneyl.

[0086] More preferably, R₄ is t-BOC, iso-butenyl, crotonyl,dimethyacryloyl, thiophenyl, thiazolyl or furyl.

[0087] In embodiments (1) and (2), R₅ is the linking group and R₆ hasthe same definition as above for R₂ for embodiments (1), (2) and (4).

[0088] In embodiment (3), R₅ has the same definition as above for R₂ forembodiments (1), (2) and (4).

[0089] In embodiment (3), R₆ has the same definition as above for R₂ forembodiments (1), (2) and (4).

[0090] In embodiment (4), R₆ is a linking group, and R₅ has the samedefinition as above for R₂ for embodiments (1), (2) and (4).

[0091] Suitable linking groups are well known in the art and includedisulfide groups, thioether groups, acid labile groups, photolabilegroups, peptidase labile groups and esterase labile groups. Preferredare disulfide groups and thioether groups.

[0092] When the linking group is a thiol- or disulfide-containing group,the side chain carrying the thiol or disulfide group can be linear orbranched, aromatic or heterocyclic. One of ordinary skill in the art canreadily identify suitable side chains.

[0093] Specific examples of the thiol- or disulfide-containingsubstituents include —(CR₁₃R₁₄)_(m)(CR₁₅R₁₆)_(n)(OCH₂CH₂)_(y)SZ,—CO(CR₁₃R₁₄)_(m)(CR₁₅R₁₆)_(n)(OCH₂CH₂)_(y)SZ,—(CR₁₃R₁₄)_(m)(CR₁₇═CR₁₈)(CR₁₅R₁₆)_(m)OCH₂CH₂)_(y)SZ, —CO—(CR₁₃R₁₄)_(m)(CR₁₇═CR₁₈)(CR₁₅R₁₆)_(m)(OCH₂CH₂)_(y)SZ,—CONR₁₂(CR₁₃R₁₄)_(m)(CR₁₅R₁₆)_(n) (OCH₂CH₂)_(y)SZ, furyl-XSZ,oxazolyl-XSZ, thiazolyl-XSZ, thiopheneyl-XSZ, imidazolyl-XSZ,morpholino-XSZ, -piperazino-XSZ, piperidino-XSZ, CO-furyl-XSZ,CO-thiopheneyl-XSZ, CO-thiazolyl-XSZ and —CO—N-methylpiperazino-XSZ,—CO-morpholino-XSZ, —CO-piperazino-XSZ, —CO-piperidino-XSZ, or—CO-N-methylpiperazino-XSZ, wherein:

[0094] Z is H or SR,

[0095] X is a linear alkyl or branched alkyl having from 1-10 carbonatoms or a polyethylene glycol spacer with 2 to 20 repeating ethyleneoxy units;

[0096] R and R₁₂ are the same or different and are linear alkyl,branched alkyl or cyclic alkyl having from 1 to 10 carbon atoms, orsimple or substituted aryl or heterocyclic, and R₁₂ can in addition beH,

[0097] R₁₃, R₁₄, R₁₅ and R₁₆ are the same or different and are H or alinear or branched alkyl having from 1 to 4 carbon atoms,

[0098] R₁₇ and R₁₈ are H or methyl,

[0099] n is an integer of 1 to 10,

[0100] m is an integer from 1 to 10 and can also be 0,

[0101] y is an integer from 1 to 20 and can also be 0.

[0102] The preferred taxanes of the first aspect of the presentinvention are those bearing a free hydroxyl group at C-10 (i.e. R₂) anda linking group at C-7 (i.e. R₅). The most preferred taxanes of thepresent invention are taxanes 2′ and 3′ shown in FIG. 3.

[0103] Embodiments (5) to (9)

[0104] In embodiments (5) to (9), R₁ is H, an electron-withdrawinggroup, such as F, NO₂, CN, Cl, CHF₂, and CF₃ or an electron donatinggroup such as —OCH₃, —OCH₂CH₃, —NR₇R₈, —OR₉. R₁′ and R¹″ are the same ordifferent and are H, an electron withdrawing group, such as F, NO₂, CN,Cl, CHF₂, and CF₃ or an electron donating group such as —OCH₃, —OCH₂CH₃,—NR₇R₈, and —OR₉.

[0105] R₇ and R₈ are the same or different and are linear, branched, orcyclic alkyl groups having 1 to 10 carbon atoms or simple or substitutedaryl. Preferably the number of carbon atoms for R₇ and R₈ is 1 to 4.Also, preferably R₇ and R₈ are the same. Examples of preferred —NR₇R₈groups include dimethyl amino, diethyl amino, dipropyl amino,di-isopropylamino and dibutyl amino, where the butyl moiety is any ofprimary, secondary, tertiary or isobutyl.

[0106] R₉ is linear, branched or cyclic alkyl having 1 to 10 carbonatoms.

[0107] Preferably, R₁ is —OCH₃, Cl, F, NO₂ and CF₃.

[0108] More preferably, R₁ is —OCH₃ and in the meta position, and one ofR₁′ and R₁″ is —OCH₃ and the other is H.

[0109] In embodiments (5), (6) and (7), R₃ and R₄ are the same ordifferent and are alkyl or alkenyl, having from 1 to 10 carbon atoms,cycloalkyl, cycloalkenyl having from 3 to 10 carbon atoms, aryl, orheterocycle and R₄ additionally is —OC(CH₃)₃ or a carbamate formed fromany of said alkyl, alkenyl, cycloalkyl, or cycloalkenyl, aryl, or anitrogen-containing heterocycle.

[0110] Preferably, one or both of R₃ and R₄ are crotonyl,dimethylacryloyl, isobutenyl, hexenyl, cyclopentenyl, cyclohexenyl,furyl, pyrollyl, thiopheneyl, thiazolyl, imidazolyl, pyridyl,morpholino, piperidino, piperazino, oxazolyl, indolyl, benzofuranyl orbenzothiopheneyl.

[0111] More preferably, one or both of R₃ and R₄ are t-BOC, iso-butenyl,crotonyl, dimethyacryloyl, thiophenyl, thiazolyl or furyl.

[0112] In embodiments (8) and (9), R₂, R₅ and R₆ are the same ordifferent and are H, a heterocyclic, or aryl ether, ester, or carbamate,or a linear, branched or cyclic alkyl or alkenyl ester or ether havingfrom 1 to 10 carbon atoms, or a carbamate of the formula —COX, wherein Xis a nitrogen-containing heterocycle, such as piperidino, morpholino,piperazino, N-methylpiperazino, or a carbamate of the formula—CONR₁₀R₁₁, wherein R₁₀ and R₁₁ are the same or different and are H,linear, branched, or cyclic alkyl having 1 to 10 carbon atoms or simpleor substituted aryl.

[0113] Preferred examples of aryl ethers, esters, and carbamates includephenyl and naphthyl.

[0114] Preferred examples of alkyl and alkenyl esters include —COCH₃,—COCH₂CH₃, crotonyl and dimethyaeryloyl. Preferred examples of alkyl andalkenyl ethers include methyl, ethyl, allyl, propyl, crotonyl anddimethyacryloyl. Preferred examples of carbamates include —CONHCH₂CH₃,—CO-morpholino, —CO-piperazino, or —CO—N-methylpiperazino.

[0115] Preferably, R₆ is H and one of R₂ and one of R₅ is H.

[0116] In embodiment (5), R₂ is the linking group, and R₅ and R₆ havethe same definition as for embodiments (8) and (9).

[0117] In embodiment (6), R₅ is the linking group, and R₂ and R₆ havethe same definition as for embodiments (8) and (9).

[0118] In embodiment (7), R₆ is the linking group or H, and R₂ and R₅have the same definition as for embodiments (8) and (9).

[0119] In embodiment (8), R₃ is the linking group, and R₄ has the samedefinition for embodiments (5), (6) and (7).

[0120] In embodiment (9), R₄ is the linking group, and R₃ has the samedefinition as for embodiments (5), (6) and (7).

[0121] Suitable linking groups are well known in the art and includedisulfide groups, thioether groups, acid labile groups, photolabilegroups, peptidase labile groups and esterase labile groups. Preferredare disulfide groups and thioether groups. When the linking group is athiol- or disulfide-containing group, the side chain carrying the thiolor disulfide group can be linear or branched alkyl, alkenyl,cycloakenyl, aromatic or heterocyclic or a polyethylene glycol. One ofordinary skill in the art can readily identify suitable side chains.

[0122] Specific examples of the thiol- or disulfide-containingsubstituents include —(CR₁₃R₁₄)_(m)(CR₁₅R₁₆)_(n)(OCH₂CH₂)_(y)SZ,—CO(CR₁₃R₁₄)_(m)(CR₁₅R₁₆)_(n)(OCH₂CH₂)_(y)SZ,—(CR₁₃R₁₄)_(m)(CR₁₇═CR₁₈)(CR₁₅R₁₆)_(m)OCH₂CH₂)_(y)SZ, —CO—(CR₁₃R₁₄)_(m)(CR₁₇═CR₁₈)(CR₁₅R₁₆)_(m)(OCH₂CH₂)_(y)SZ,—CONR₁₂(CR₁₃R₁₄)_(m)(CR₁₅R₁₆)_(n) (OCH₂CH₂)_(y)SZ, furyl-XSZ,oxazolyl-XSZ, thiazolyl-XSZ, thiopheneyl-XSZ, imidazolyl-XSZ,morpholino-XSZ, -piperazino-XSZ, piperidino-XSZ, CO-furyl-XSZ,CO-thiopheneyl-XSZ, CO-thiazolyl-XSZ and —CO-N-methylpiperazino-XSZ,—CO-morpholino-XSZ, —CO-piperazino-XSZ, —CO-piperidino-XSZ, or—CO—N-methylpiperazino-XSZ, wherein:

[0123] Z is H or SR,

[0124] X is a linear alkyl or branched alkyl having from 1-10 carbonatoms or a polyethylene glycol spacer with 2 to 20 repeating ethyleneoxy units;

[0125] R and R₁₂ are the same or different and are linear alkyl,branched alkyl or cyclic alkyl having from 1 to 10 carbon atoms, orsimple or substituted aryl or heterocyclic, and R₁₂ can in addition beH,

[0126] R₁₃, R₁₄, R₁₅ and R₁₆ are the same or different and are H or alinear or branched alkyl having from 1 to 4 carbon atoms,

[0127] R₁₇ and R₁₈ are H or methyl,

[0128] n is an integer of 1 to 10,

[0129] m is an integer from 1 to 10 and can also be 0,

[0130] y is an integer from 1 to 20 and can also be 0.

[0131] The taxanes of the present invention can be synthesized accordingto known methods. The starting material for the synthesis is thecommercially available 10-deacetylbaccatin III, shown in FIG. 7. Thechemistry to introduce various substituents is described in severalpublications (Ojima et al, J. Med. Chem. 39: 3889-3896 (1996), Ojima etal., J. Med. Chem. 40: 267-278 (1997); I. Ojima et al., Proc. Natl.Acad. Sci., 96: 4256-4261 (1999); I. Ojima et al., U.S. Pat. Nos.5,475,011 and 5,811,452). The preparation of representative taxanes ofthe present invention is described in the Examples below.

[0132] The substituent R₁ on the phenyl ring and the position of thesubstituent R₁ can be varied until a compound of the desired toxicity isobtained. Furthermore, the degree of substitution on the phenyl ring canbe varied to achieve a desired toxicity. That is, the phenyl ring canhave one or more substituents (e.g., mono-, di-, or tri-substitution ofthe phenyl ring) which provide another means for achieving a desiredtoxicity. High cytotoxicity is defined as exhibiting a toxicity havingan IC₅₀ in the range of 1×10⁻¹² to 3×10⁻⁹ M, when measured in vitro withcultured cancer cells upon a 72 hour exposure time to the drug. One ofordinary skill in the art can determine the appropriate chemical moietyfor R₁ and the appropriate position for R₁ using only routineexperimentation.

[0133] For example electron donating groups at the meta position areexpected to increase the cytotoxic potency, while substitution at thepara position is not expected to increase the potency as compared to theparent taxane. Typically a few representative taxanes with substituentsat the different positions (ortho, meta and para) will be preparedinitially and evaluated for in vitro cytotoxicity.

[0134] The new taxoids described in FIGS. 5 and 16 can be prepared bythe β-lactam synthon method (Ojima, I.; Habus, I.; Zhao, M.; Zucco, M.;Park, Y. H.; Sun, C. M.; Brigaud, T. Tetrahedron, 48: 6985 (1992);Holton, R. A.; Biediger, R. J.; Boatman, P. D. in Taxol: Science andApplications; Suffness, M., Ed.; CRC: Boca Raton, 1995, p. 97) usingappropriately derivatized baccatin III analog (7) and β-lactams asstarting materials. The β-lactams 4-6 d, 19-25 and 38-44 can be preparedby previously described methods (Brieva, R. Crich, J. Z.; Sih, C. J. J.Org. Chem., 58: 1068 (1993); Palomo, C.; Arrieta, A.; Cossio, F.;Aizpurua, J. M.; Mielgo, A.; Aurrekoetxea, N. Tetrahedron Lett., 1990,31, 6429) the baccatin III analog (7) can be prepared using thecommercially available 10-deacetylbaccatin III (10-DAB) (FIG. 7) asstarting material.

[0135] The β-lactams 6 a-d, 21-25 and 40-44 can be coupled with thebaccatin III analog (7) in the presence of NaH or LiHMDS to giveprotected taxoids 8-11, 26-30 and 45-49. The silyl protecting groups canbe finally deprotected in the presence of HF-pyridine to yield thedesired taxanes 12-15, 31-35, and 50-54 (FIGS. 16a, 16 b and 16 c).

[0136] Disulfide-containing taxoids of the present invention (FIGS. 6,17) can be synthesized from the intermediates described above (8-11,26-30, 45-49). The C-10 acetate can be removed successfully withhydrazine monohydrate. The reesterification of the C-10 position canthen be carried out in the presence of EDC(1-[3-(dimethylamino)propyl-3-ethylcarbodiimide hydrochloride) employingthe carbodiimide based coupling protocol using the required disulfidederivatives of the carboxylic acids. The coupled products can bedeprotected with HF-pyridine to give the desired disulfide-containingtaxoids (FIGS. 17a and 17 b).

[0137] The disulfide or thiol-containing substituent can also beintroduced at one of the other positions where a hydroxyl group alreadyexists. The chemistry to protect the various hydroxyl groups, whilereacting the desired one, has been described previously (see, forexample, the references cited, supra). The substituent is introduced bysimply converting the free hydroxyl group to a disulfide-containingether, a disulfide-containing ester, or a disulfide-containingcarbamate. Alternatively, a polyethylene glycol spacer may be introducedbetween the disulfide or thiol substituent and the hydroxy group that isbeing derivatized. (See, for example, U.S. Ser. No. 10/144,042, filedMay 14, 2002) This transformation is achieved as follows. The desiredhydroxyl group is deprotonated by treatment with the commerciallyavailable reagent lithium hexamethyldisilazane (1.2 equivalents) intetrahydrofuran at −40° C. as described in I. Ojima et al, supra. Theresulting alkoxide anion is then reacted with an excess of a dihalocompound, such as dibromoethane, to give a halo ether. Displacement ofthe halogen with a thiol (by reaction with potassium thioacetate andtreatment with mild base or hydroxylamine) will provide the desiredthiol-containing taxane. The thiol group can be converted into a methylor pyridyl disulfide by reaction with methyl methane thiolsulfonate ordithiodipyridine respectively. This method is described in U.S. Pat. No.5,416,064.

[0138] The desired hydroxyl group can also be esterified directly byreaction with an acyl halide, such as 3-bromopropionyl chloride to givea bromo ester. Displacement of the bromo group by treatment withpotassium thioacetate and further processing as described above willprovide the thiol or disulfide-containing taxane ester. In order toprepare disulfide-containing carbamates, the hydroxyl group can bereacted with a commercially available chloroformate, such aspara-nitrophenyl chloroformate, followed by reaction with an amino alkyldisulfide (e.g., methyldithio cysteamine).

[0139] Alternatively, the thiol or disulfide substituent can beincorporated into the β-lactam subunit, which is then reacted with theappropriately protected 10-deacetylbaccatin III to give the desiredtaxanes bearing a thiol or disulfide linking group at the C-3′ position.

[0140] The new taxanes and the disulfide containing taxane drugs of theinvention can be evaluated for their ability to suppress proliferationof human tumor cell lines in vitro. The human tumor cell lines A-549(human lung carcinoma) and MCF-7 (human breast tumor, are used for theassessment of cytotoxicity of these compounds. Cells are exposed to thecompounds for 72 hours and the surviving fractions of cells are measuredin direct plating efficiency assays as previously described (Goldmacheret al, J. Cell. Biol. 102: 1312-1319 (1986) and IC₅₀ values are thencalculated from this data.

[0141] The results of the in vitro cytotoxicity measurement of taxoidsand disulfide-containing taxoids according to the second aspect of thepresent invention are shown in FIGS. 18 and 19. FIG. 18 shows theresults of the cytotoxicity determination of twelve new taxanes of thepresent invention. Except for taxane 52, which bears a phenylsubstituent at R₄, all the other new taxanes were extremely potenttowards both A-549 and MCF-7 cell lines with IC₅₀ values in the 10⁻¹⁰ to10⁻¹¹ M range. Taxane 52 was less cytotoxic with an IC₅₀ value of 3×10⁻⁹M towards both cell lines that were tested. Similarly,disulfide-containing taxoids of the present invention are also extremelypotent toward both A-549 and MCF-7 cells and display steep killingcurves (FIG. 19).

[0142] The effectiveness of the compounds of the invention astherapeutic agents depends on the careful selection of an appropriatecell-binding agent. Cell-binding agents may be of any kind presentlyknown, or that become known and include peptides and non-peptides.Generally, these can be antibodies, or fragments thereof, (especiallymonoclonal antibodies), lymphokines, hormones, growth factors, vitamins,nutrient-transport molecules (such as transferrin), or any othercell-binding molecule or substance.

[0143] More specific examples of cell-binding agents that can be usedinclude:

[0144] fragments of antibodies such as sFv, Fab, Fab′, and F(ab′)₂(Parham, J. Immunol.131: 2895-2902 (1983); Spring et al, J. Immunol.113: 470-478 (1974); Nisonoff et al, Arch. Biochem. Biophys. 89: 230-244(1960));

[0145] interferons (e.g. α, β, γ);

[0146] lymphokines such as IL-2, IL-3, IL-4, IL-6;

[0147] hormones such as insulin, TRH (thyrotropin releasing hormones),MSH (melanocyte-stimulating hormone), steroid hormones, such asandrogens and estrogens;

[0148] vitamins such as folic acid;

[0149] growth factors and colony-stimulating factors such as EGF, TGF-α,G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5: 155-158 (1984));and

[0150] transferrin (O'Keefe et al, J. Biol. Chem. 260: 932-937 (1985)).

[0151] Monoclonal antibody techniques allow for the production ofextremely specific cell-binding agents in the form of specificmonoclonal antibodies or fragments thereof. Particularly well known inthe art are techniques for creating monoclonal antibodies, or fragmentsthereof, by immunizing mice, rats, hamsters, or any other mammal withthe antigen of interest such as the intact target cell, antigensisolated from the target cell, whole virus, attenuated whole virus, andviral proteins such as viral coat proteins. Sensitized human cells canalso be used. Another method of creating monoclonal antibodies, orfragments thereof, is the use of phage libraries of sFv (single chainvariable region), specifically human sFv. (See e.g., Griffiths et al.,U.S. Pat. No. 5,885,793; McCafferty et al., WO 92/01047; Liming et al.,WO 99/06587.)

[0152] Selection of the appropriate cell-binding agent is a matter ofchoice that depends upon the particular cell population to be targeted,but in general monoclonal antibodies are preferred if an appropriate oneis available.

[0153] For example, the monoclonal antibody MY9 is a murine IgG₁antibody that binds specifically to the CD33 antigen (J. D. Griffin etal Leukemia Res., 8: 521 (1984)) which can be used if the target cellsexpress CD33, such as in the disease of acute myelogenous leukemia(AML). Similarly, the monoclonal antibody anti-B4 is a murine IgG₁ thatbinds to the CD19 antigen on B cells (Nadleret al, J. Immunol. 131:244-250 (1983)) and can be used if the target cells are B cells ordiseased cells that express this antigen, such as in non-Hodgkin'slymphoma or chronic lymphoblastic leukemia. Similarly, the antibody N901is a murine monoclonal IgG₁ antibody that binds to CD56 found on smallcell lung carcinoma cells and on cells of other tumors of neuroendocrineorigin (Roy et al. J. Nat. Cancer Inst. 88:1136-1145 (1996)).

[0154] Antibodies that target solid tumors are also useful, such as theC242 antibody which binds to a carbohydrate antigen found on MUC1present on pancreatic and colorectal tumors. (U.S. Pat. No. 5,552,293);antibody J591, which binds to PSMA (prostate specific membrane antigen)which is expressed on prostate cancer cells and on endothelial cells ofneovasculature in tumors (U.S. Pat. No. 6,107,090, He Liu et al. CancerRes. 57: 3629-3634 (1997); and antibodies to HER-2, which isoverexpressed on certain breast tumors. Examples of anti-HER-2antibodies are the TA1 antibody (L. A. Maier et al. Cancer Res. 51:5361-5369 (1991)) and the 4D5 antibody (U.S. Pat. Nos. 6,387,371 and6,399,063).

[0155] Additionally, GM-CSF, which binds to myeloid cells, can be usedas a cell-binding agent to diseased cells from acute myelogenousleukemia. IL-2, which binds to activated T-cells, can be used forprevention of transplant graft rejection, for therapy and prevention ofgraft-versus-host disease, and for treatment of acute T-cell leukemia.MSH, which binds to melanocytes can be used for the treatment ofmelanoma. Folic acid, which targets the folate receptor expressed onovarian and other cancers, is also a suitable cell-binding agent.

[0156] Cancers of the breast and testes can be successfully targetedwith estrogen (or estrogen analogues) or androgen (or androgenanalogues), respectively, as cell-binding agents.

[0157] Conjugates of the taxanes of the invention and a cell-bindingagent can be formed using any techniques presently known or laterdeveloped. Numerous methods of conjugation are taught in U.S. Pat. Nos.5,416,064 and 5,475,092. The taxane ester can be modified to yield afree amino group and then linked to an antibody or other cell-bindingagent via an acid labile linker or a photolabile linker. The taxaneester can be condensed with a peptide and subsequently linked to acell-binding agent to produce a peptidase labile linker. The hydroxylgroup on the taxane ester can be succinylated and linked to acell-binding agent to produce a conjugate that can be cleaved byintracellular esterases to liberate free drug. Most preferably, thetaxane ethers, esters, or carbamates are treated to create a free orprotected thiol group, and then the disulfide- or thiol-containingtaxanes are linked to the cell-binding agent via disulfide bonds.

[0158] Representative conjugates of the invention are antibody-taxane,antibody fragment-taxane epidermal growth factor (EGF)-taxane,melanocyte stimulating hormone (MSH)-taxane, thyroid stimulating hormone(TSH)-taxane, estrogen-taxane, estrogen analogue-taxane,androgen-taxane, androgen analogue-taxane, and folate-taxane.

[0159] Taxane conjugates of antibodies, antibody fragments, protein orpeptide hormones, protein or peptide growth factors and other proteinsare made in the same way by known methods. For example, peptides andantibodies can be modified with cross linking reagents such asN-succinimidyl 3-(2-pyridyldithio)propionate, N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP),4-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl dithio)-toluene (SMPT),N-succinimidyl-3-(2-pyridyldithio) butyrate (SDPB), 2-iminothiolane, orS-acetylsuccinic anhydride by known methods. See, Carlsson et al,Biochem. J. 173: 723-737 (1978); Blattler et al, Biochem. 24: 1517-1524(1985); Lambert et al, Biochem. 22: 3913-3920 (1983); Klotz et al, Arch.Biochem. Biophys. 96: 605 (1962); and Liu et al, Biochem. 18: 690(1979), Blakey and Thorpe, Antibody, Immunoconjugates &Radiopharmaceuticals, 1: 1-16 (1988), Worrell et al Anti-Cancer DrugDesign 1: 179-184 (1986). The free or protected thiol-containingcell-binding agent thus derived is then reacted with a disulfide- orthiol-containing taxane to produce conjugates. The conjugates can bepurified by HPLC or by gel filtration.

[0160] Similarly, for example, estrogen and androgen cell-binding agentssuch as estradiol and and rostanediol can be esterified at the C-17hydroxy group with an appropriate disulfide containing carboxylic acidusing e.g., dicyclohexylcarbodiimide as a condensing agent. Examples ofsuch carboxylic acids that can be employed are 3-(2-pyridyldithio)propanoic acid, 3-methyldithiopropanoic acid, 4-(2-pyridyldithio)pentanoic acid, and 3-phenyldithiopropanoic acid. Esterification of theC-17 hydroxy group can also be achieved by reaction with anappropriately protected thiol group containing carboxylic acid chloridesuch as 3-S-acetylpropanoyl chloride. Other methods of esterificationcan also be employed as described in the literature (Haslam, Tetrahedron36: 2409-2433 (1980)). The protected or free thiol-containing androgenor estrogen can then be reacted with a disulfide- or thiol-containingtaxane to produce a conjugate. The conjugate can be purified by columnchromatography on silica gel or by HPLC. Folic acid can be condensedwith a suitable hydrazide such as 4-(2-pyridyldithio) pentanoic acidhydrazide in the presence of a condensing agent such as dicyclohexylcarbodiimide to give a hydrazone containing an active disulfide. Thedisulfide-containing folate can then be reacted with a thiol-containingtaxane to produce a conjugate that can be purified by columnchromatography over silica gel or by HPLC

[0161] Preferably, monoclonal antibody- or cell-binding agent-taxaneconjugates are those that are joined via a disulfide bond, as describedabove, that are capable of delivering taxane molecules. Suchcell-binding conjugates are prepared by known methods such as bymodifying monoclonal antibodies with succinimidylpyridyl-dithiopropionate (SPDP) (Carlsson et al, Biochem. J. 173:723-737 (1978)). The resulting thiopyridyl group is then displaced bytreatment with thiol-containing taxanes to produce disulfide linkedconjugates. Alternatively, in the case of the aryldithio-taxanes, theformation of the cell-binding conjugate is effected by directdisplacement of the aryl-thiol of the taxane by sulfhydryl groupspreviously introduced into antibody molecules. Conjugates containing 1to 10 taxane drugs linked via a disulfide bridge are readily prepared byeither method.

[0162] More specifically, a solution of the dithiopyridyl modifiedantibody at a concentration of 1 mg/ml in 0.1 M potassium phosphatebuffer, at pH 6.5 containing 1 mM EDTA is treated with thethiol-containing taxane (1.25 molar eq./dithiopyridyl group). Therelease of thiopyridine from the modified antibody is monitoredspectrophotometrically at 343 nm and is complete in about 20 hours. Theantibody-taxane conjugate is purified and freed of unreacted drug andother low molecular weight material by gel filtration through a columnof Sephadex G-25 or Sephacryl S300. The number of taxane moieties boundper antibody molecule can be determined by measuring the ratio of theabsorbance at 230 nm and 275 nm. An average of 1 to 10 taxanemolecules/antibody molecule can be linked via disulfide bonds by thismethod.

[0163] Antibody-taxane conjugates with non-cleavable links can also beprepared. The antibody can be modified with crosslinking reagents suchas N-sucinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC),sulfo-SMCC, N-succinimidyl 4-maleimidobutyrate (SMB), sulfo-SMB,N-succinimidyl 6-maleimidocaproate (SMC), sulfo-SMC, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS orsuccinimidyl-iodoacetate, as described in the literature, to introduce1-10 reactive groups. See, Yoshitake et al, Eur. J. Biochem. 101:395-399 (1979); Hashida et al, J. Applied Biochem. 6: 56-63 (1984); andLiu et al, Biochem. 18: 690-697 (1979). The modified antibody is thenreacted with the thiol-containing taxane derivative to produce aconjugate. The conjugate can be purified by gel filtration through aSephadex G-25 column.

[0164] The modified antibodies, or fragments thereof, are treated withthe thiol-containing taxanes (1.25 molar equivalent/maleimido group).The mixtures are incubated overnight at about 4° C. The antibody-taxaneconjugates are purified by gel filtration through a Sephadex G-25column. Typically, an average of 1 to 10 taxanes is linked per antibody.

[0165] A preferred method is to modify antibodies, or fragments thereof,with succinimidyl-4-(maleimidomethyl)-cyclohexane-1-carboxylate (SMCC)to introduce maleimido groups followed by reaction of the modifiedantibody or fragment with the thiol-containing taxanes to give athioether linked conjugate. Again, conjugates with 1 to 10 drugmolecules per antibody molecule result.

[0166] Cytotoxicity of antibody conjugates of these taxoids tonon-adherent cell lines such as Namalwa and HL-60 can be measured byback-extrapolation of cell proliferation curves as described inGoldmacher et al, J. Immunol. 135: 3648-3651 (1985). Cytotoxicity ofthese compounds to adherent cell lines such as SKBR3 and A431 can bedetermined by clonogenic assays as described in Goldmacher et al, J.Cell Biol. 102: 1312-1319 (1986).

[0167] The present invention also provides a therapeutic compositioncomprising:

[0168] (a) an effective amount of one or more taxanes linked to acell-binding agent, and

[0169] (b) a pharmaceutically acceptable carrier, diluent, or excipient.

[0170] Similarly, the present invention provides a method for inducingcell death in selected cell populations comprising contacting targetcells or tissue containing target cells with an effective amount of acytotoxic agent comprising one or more taxanes linked to a cell-bindingagent.

[0171] The cytotoxic agent is prepared as described above.

[0172] Suitable pharmaceutically acceptable carriers, diluents, andexcipients are well known and can be determined by those of ordinaryskill in the art as the clinical situation warrants.

[0173] Examples of suitable carriers, diluents and/or excipientsinclude: (1) Dulbecco's phosphate buffered saline, pH about 7.4,containing or not containing about 1 mg/ml to 25 mg/ml human serumalbumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose; andmay also contain an antioxidant such as tryptamine and a stabilizingagent such as Tween 20.

[0174] The method for inducing cell death in selected cell populationscan be practiced in vitro, in vivo, or ex vivo.

[0175] Examples of in vitro uses include treatments of autologous bonemarrow prior to their transplant into the same patient in order to killdiseased or malignant cells: treatments of bone marrow prior to theirtransplantation in order to kill competent T cells and preventgraft-versus-host-disease (GVHD); treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen.

[0176] The conditions of non-clinical in vitro use are readilydetermined by one of ordinary skill in the art.

[0177] Examples of clinical ex vivo use are to remove tumor cells orlymphoid cells from bone marrow prior to autologous transplantation incancer treatment or in treatment of autoimmune disease, or to remove Tcells and other lymphoid cells from autologous or allogenic bone marrowor tissue prior to transplant in order to prevent GVHD. Treatment can becarried out as follows. Bone marrow is harvested from the patient orother individual and then incubated in medium containing serum to whichis added the cytotoxic agent of the invention, concentrations range fromabout 10 μM to 1 μM, for about 30 minutes to about 48 hours at about 37°C. The exact conditions of concentration and time of incubation, i.e.,the dose, are readily determined by one of ordinary skill in the art.After incubation the bone marrow cells are washed with medium containingserum and returned to the patient intravenously according to knownmethods. In circumstances where the patient receives other treatmentsuch as a course of ablative chemotherapy or total-body irradiationbetween the time of harvest of the marrow and reinfusion of the treatedcells, the treated marrow cells are stored frozen in liquid nitrogenusing standard medical equipment.

[0178] For clinical in vivo use, the cytotoxic agent of the inventionwill be supplied as a solution or a lyophilized powder that are testedfor sterility and for endotoxin levels. Examples of suitable protocolsof conjugate administration are as follows. Conjugates are given weeklyfor 4 weeks as an intravenous bolus each week. Bolus doses are given in50 to 100 ml of normal saline to which 5 to 10 ml of human serum albumincan be added. Dosages will be 10 μg to 2000 mg per administration,intravenously (range of 100 ng to 20 mg/kg per day). After four weeks oftreatment, the patient can continue to receive treatment on a weeklybasis. Specific clinical protocols with regard to route ofadministration, excipients, diluents, dosages, times, etc., can bedetermined by one of ordinary skill in the art as the clinical situationwarrants.

[0179] Examples of medical conditions that can be treated according tothe in vivo or ex vivo methods of inducing cell death in selected cellpopulations include malignancy of any type including, for example,cancer of the lung, breast, colon, prostate, kidney, pancreas, ovary,and lymphatic organs; autoimmune diseases, such as systemic lupus,rheumatoid arthritis, and multiple sclerosis; graft rejections, such asrenal transplant rejection, liver transplant rejection, lung transplantrejection, cardiac transplant rejection, and bone marrow transplantrejection; graft versus host disease; viral infections, such as CMVinfection, HIV infection, AIDS, etc.; and parasite infections, such asgiardiasis, amoebiasis, schistosomiasis, and others as determined by oneof ordinary skill in the art.

EXAMPLES

[0180] The invention will now be illustrated by reference tonon-limiting examples. Unless otherwise stated, all percents, ratios,parts, etc. are by weight.

Example 1

[0181] Preparation of Taxane2′

[0182] Taxane2′(3′-dephenyl-3′-(isobutenyl)-7-(methyldisulfonyl-propanoyl)-docetaxel)was prepared from commercially available 10-deacetylbaccatin III (FIG.7) following the scheme shown in FIG. 8.

[0183] Compounds 4-6′ were prepared as described by Greene et al. in J.Am. Chem. Soc. 110: 5917-5919 (1988) and by Ojima et al, J. Med. Chem.39: 3889-3896 (1996) and references cited therein.

[0184] Compound7′(7-(triethylsilyl)-2′-(triisopropylsilyloxy)-3′-dephenyl-3′-(isobutenyl)-docetaxel)was prepared by adding hydrazine monohydrate (1 mL) to a solution of 6′(65 mg, 0.059 mmol) in ethanol (2 mL) at room temperature. The reactionwas stirred at room temperature and monitored by thin layerchromatography using 40% ethyl acetate in hexane. After 1 hour thereaction was complete by thin layer chromatography and quenched withsaturated aqueous ammonium chloride (10 mL). The aqueous layer wasextracted with ethyl acetate (10 ml×3). The combined extracts were driedover anhydrous magnesium sulfate and concentrated in vacuo. The residuewas purified on a silica gel column using 40% ethyl acetate in hexane asthe eluant to afford product 7′ as a white solid (42 mg, 69%): 1H NMR(CDCl3) δ0.53 (m, 6 H), 0.92 (t, J=8.0 Hz, 9 H), 1.11 (in, 24 H), 1.20(s, 3 H), 1.23 (s, 3 H), 1.32 (s, 9 H), 1.71 (s, 3 H), 1.72 (m, 3 H),1.78 (s, 3 H), 1.92 (m, 4 H), 2.35 (m, 5 H), 3.89 (d, J=6.8 Hz, 1 H),4.18 (d, J=8.4 Hz, 1 H), 4.23 (d, J=2.0 Hz, 1 H), 4.28 (d, J=8.4 Hz, 1H), 4.37 (dd, J=6.4, 10.4 Hz, 1 H), 4.41 (d, J=3.2 Hz, 1 H), 4.80 (m, 2H), 4.91 (d, J=8.0 Hz, 1 H), 5.10 (d, J=2.0 Hz, 1 H), 5.31(d, J=8.8 Hz,1 H), 5.63(d, J=7.2Hz, 1 H), 6.13 (t, J=9.0Hz, 1 H), 7.43 (t, J=8.0 Hz,2 H), 7.57 (t, J=8.0 Hz, 1 H), 8.07 (d, J=8.0 Hz 2 H). m/z LC/MS forC56H89NO14Si2Na+: calcd: 1078.58; found: 1078.40.

[0185] Compound 8′(2′-(triisopropylsilyloxy)-3′-dephenyl-3′-(isobutenyl)-docetaxel) wasprepared by the following steps. A solution of compound 7′ (35 mg, 0.029mmol) was made by adding a solution of 0.1 N HCl in ethanol (5 mL) at 0oC. The solution was stirred with gradual warming to room temperatureand allowed to stir for 16 hrs. The reaction was quenched with saturatedaqueous sodium bicarbonate (10 mL), and the aqueous layer was extractedwith ethyl acetate (15 ml×3). The combined extracts were dried overanhydrous magnesium sulfate and concentrated in vacuo. The residue waspurified on a silica gel column using 50% ethyl acetate in hexane as theeluant to afford product 8′ as a white solid (20 mg, 64%): δ1.11 (m, 24H), 1.23 (s, 3 H), 1.26 (s, 3 H), 1.30 (s, 9 H), 1.74 (s, 6 H), 1.79 (s,3 H), 1.84 (m, 1 H), 1.92 (s, 3 H), 2.36 (s, 3 H), 2.38 (m, 1 H), 2.57(m, 1 H), 3.92 (d, J=6.8 Hz, 1 H), 4.17 (d, J=1.2 Hz, 1 H), 4.22 (d,J=8.0 Hz, 1 H), 4.23 (m, 1 H), 4.31 (d, J=8.0Hz, 1 H), 4.42 (d, J=2.8Hz, 1 H), 4.75 (m, 1 H), 4.85 (m, 1 H), 4.95 (d, J=7.6 Hz, 1 H), 5.20(s, 1 H), 5.33 (d, J=8.8 Hz, 1 H), 5.68 (d, J=7.2 Hz, 1 H), 6.14 (t,J=8.8 Hz, 1 H), 7.46 (t, J=8.0 Hz, 2 H), 7.60 (t, J=8.0 Hz, 1 H), 8.10(d, J=8.0 Hz 1 H).

[0186] Compound 9′(2′-(triisopropylsilyloxy)-3′-dephenyl-3′-(isobutenyl)-7-(methyldisulfamyl-propanoyl)-docetaxel)was prepared by the following steps. To a solution of 8′ (20 mg, 0.020mmol) in methylene chloride (3 mL) was added DMAP (3 mg, 0.02 mmol),dithio acid (3 mg, 0.018 mmol) and EDC (8 mg, 0.042 mmol). The resultingmixture was stirred overnight. Thin layer chromatography analysis using25% ethyl acetate in hexanes revealed virtually all starting materialwas consumed and a new spot was present. The reaction was quenched withsaturated aqueous ammonium chloride (10 mL) and extracted into methylenechloride (10 ml×3). The combined extracts were dried over anhydrousmagnesium sulfate and concentrated in vacuo. The residue was purified ona silica gel column using 25% ethyl acetate in hexane as the eluant toafford 9′ as a white solid (9 mg, 41%): 1H NMR (CDCl3) δ1.11 (m, 24 H),1.22 (s, 3 H), 1.34 (s, 9 H), 1.76 (s, 3 H), 1.80 (s, 3 H), 1.85 (s, 3H), 1.95 (m, 4 H), 2.36 (s, 3 H), 2.41 (m, 1 H), 2.42 (s, 3 H), 2.54 (m,1 H), 2.70 (t, J=7.2 Hz, 2 H), 2.88 (m, 2 H), 3.93 (br s, 1 H), 4.04 (d,J=7.2 Hz, 1 H), 4.24 (d, J=8.8 Hz, 1 H), 4.33 (d, J=8.8 Hz, 1 H), 4.43(d, J=2.8 Hz, 1 H), 4.77 (m, 1 H), 4.86 (m, 1 H), 4.94 (d, J=8.0 Hz, 1H), 5.32 (m, 2 H), 5.54 (dd, J=6.8, 10.4 Hz, 1 H), 5.69 (d, J=7.2 Hz, 1H), 6.13 (t, J=8.8 Hz, 1 H), 7.47 (t, J=8.0 Hz, 2 H), 7.61 (t, J=8.0 Hz,1 H), 8.10 (d, J=8.0 Hz 1 H). m/z LC/MS for C54H81NO15S2SiNa+: calcd:1098.48; found: 1098.28.

[0187] Taxane 2′(3′-dephenyl-3′-(isobutenyl)-7-(methyldisulfonyl-propanoyl)-docetaxel)was prepared by the following steps. To a solution of 9′ (9 mg, 0.008mmol) in pyridine-acetonitrile (1/1, 2 mL) was added HF/pyridine (70:30,0.1 mL) at 0 oC., and the mixture was stirred for 24 hours with warmingto room temperature. The reaction was quenched with saturated aqueoussodium bicarbonate. The reaction mixture was then diluted with ethylacetate (5 mL×2), the combined organic layers were washed with water (5mL), dried over anhydrous sodium sulfate and concentrated in vacuo. Theresidue was purified on a silica gel column using 60% ethyl acetate inhexane as the eluant to afford the final product 2′ as a white solid (5mg, 64%): 1H NMR (CDC13) δ1.10 (s, 3 H), 1.21 (s, 3 H), 1.36 (s, 9 H),1.56 (s, 3 H), 1.77 (s, 6 H), 1.86 (s, 3 H), 1.94 (m, 1 H), 1.97 (s, 3H), 2.35 (m, 1H), 2.37 (s, 3 H), 2.42 (s, 3 H), 2.56 (m, 1 H), 2.70 (t,J=7.2 Hz, 2 H), 2.88 (dd, J=2.4, 6.8 Hz, 2 H), 3.36 (br d, J=4.8 Hz , 1H), 3.95 (d, J=3.2 Hz, 1 H), 4.01 (d, J=6.8 Hz, 1 H), 4.23 (in, 2 H),4.33 (d, J=8.4 Hz, 1 H), 4.77 (m, 2 H), 4.94 (d, J=7.6 Hz, 1 H), 5.31(m, 1 H), 5.32 (d, J=1.6 Hz, 1 H), 5.51 (dd, J=7.2, 10.8 Hz, 1 H), 5.68(d, J=7.2 Hz, 1 H), 6.16 (t, J=9.0 Hz, 1 H), 7.48 (t, J=8.0 Hz, 2 H),7.62 (t, J=8.0 Hz, 1 H), 8.11 (d, J=8.0 Hz 1 H). m/z LC/MS forC45H61NO15S2Na+: calcd: 942.35; found: 942.47.

Example 2

[0188] Preparation of Taxne 3′

[0189] Taxane3′(3′-dephenyl-3′-(isobutenyl)-2-debenzoyl-2-(2,5-dimethoxybenzoyl)-7-(methyldisulfonyl-propanoyl)-docetaxel)was prepared from compound 10′ following the scheme shown in FIG. 9.

[0190] Compound 10′ (7-(Triethylsilyl)-2′-(triisopropylsilyloxy)-3′-dephenyl-3′-(isobutenyl)-2-debenzoyl-2-(2,5-dimethoxybenzoyl)-docetaxel)was prepared by the following steps. To a solution of 9′ (36 mg, 0.031mmol) in ethanol (1.5 mL) was added hydrazine monohydrate (1 mL) at roomtemperature. The reaction was stirred at room temperature and monitoredby thin layer chromatography using 40% ethyl acetate in hexane(developed twice). After 1 hour the reaction was complete by thin layerchromatography and quenched with saturated aqueous ammonium chloride (10mL). The aqueous layer was extracted with ethyl acetate (10 ml×3). Thecombined extracts were dried over anhydrous magnesium sulfate andconcentrated in vacuo. The residue was purified on a silica gel columnusing 35% ethyl acetate in hexane as the eluant to afford thedeacetylated product 10′ as a white solid (19 mg, 57%): 1H NMR (CDC13)δ0.56 (m, 6 H), 0.92 (t, J=8.0 Hz, 9 H), 1.11 (m, 27 H), 1.22 (s, 3 H),1.23 (s, 3 H), 1.38 (m, 10 H), 1.69 (s, 3 H), 1.72 (m, 3 H), 1.78 (s, 3H), 1.89 (s, 3 H), 1.93 (m, 1 H), 2.18 (s, 3 H), 2.32 (m, 1 H), 2.44 (m,2 H), 3.81 (s, 3 H), 3.82 (d, J=6.8 Hz, 1 H), 3.96 (s, 3 H), 4.25 (d,J=2.0 Hz, 1 H), 4.29 (d, J=8.0 Hz, 1 H), 4.34 (dd, J=6.4, 10.4 Hz, 1 H),4.39 (d, J=2.0 Hz, 1 H), 4.42 (d, J=8.0 Hz, 1 H), 4.76 (t, J=9.2 Hz, 1H), 4.89 (m, 2 H), 5.11 (d, J=2.0 Hz, 1 H), 5.34 (d, J=8.8 Hz, 1 H),5.64 (d, J=6.4 Hz, 1 H), 6.13 (t, J=9.0 Hz, 1 H), 6.94 (d, J=9.2 Hz, 1H), 7.06 (dd, J=9.2, 3.2 Hz, 1 H), 7.29 (d, J=2.8 Hz 1 H). m/z LC/MS forC58H93NO16Si2Na+: calcd: 1138.60; found: 1138.43.

[0191] Compound 11′(2′-(triisopropylsilyoxy)-3′-dephenyl-3′-(isobutenyl)-2-debenzoyl-2-(2,5-dimethoxybenzoyl)-docetaxel)was prepared by the following steps. A solution of 5% hydrochloric acidin ethanol (9.0 mL) was added to 10′(86.4 mg, 0.0774 mmol) at 0 oC. Themixture was stirred under N2, warming to room temperature. After 5 hoursthe reaction was quenched with saturated aqueous sodium bicarbonate andextracted into ethyl acetate (25 mL×2). The combined ethyl acetatelayers were then washed with water (25 mL×2), dried over anhydrousmagnesium sulfate and concentrated in vacuo. The crude residue waspurified on a silica gel column with 50% ethyl acetate in hexanes as theeluant. Product 11′ was isolated as a white solid (61.5 mg, 79%): 1H NMR(CDCl3) δ1.08 (s, 27 H), 1.23 (s, 3H), 1.36 (s, 9 H), 1.58 (m, 1 H),1.67 (s, 3 H), 1.70 (s, 3 H), 1.76 (s, 3 H), 1.82 (m, 2 H), 1.88 (s, 3H), 2.16 (s, 3 H), 2.31 (m, 1 H), 2.50 (m, 2 H), 3.17 (br s, 1 H), 3.79(s, 3 H), 3.85 (d, J=6.4 Hz, 1 H), 3.95 (s, 1 H), 4.18 (m, 2 H), 4.29(d, J=8.4 Hz, 1 H), 4.37 (d, J=2 Hz, 1 H), 4.41 (d, J=8.4 Hz, 1 H), 4.74(t, J=9 Hz, 1 H), 4.90 (t, J=9.8 Hz, 2 H), 5.17 (d, J=1.6 Hz, 1 H), 5.32(d, J=9.2 Hz, 1 H), 5.65 (d, J=6.8 Hz, 1 H), 6.10 (t, J=8.8 Hz, 1 H),6.93 (d, J=9.2 Hz, 1 H), 7.05 (dd, J=9.2, 3.0 Hz, 1 H), 7.28 (d, J=3.0Hz, 1 H). m/z LC/MS for C52H79NO16SiNa+: calcd: 1024.52; found: 1024.31.

[0192] Compound 12′(2′-(triisopropylsilyoxy)-3′-dephenyl-3′-(isobutenyl)-2-debenzoyl-2-(2,5-dimethoxybenzoyl)-7-(methyldisulfonyl-propanoyl)-docetaxel)was prepared by the following steps. To a solution of 11′ (25 mg, 0.025mmol), EDC (10 mg, 0.05 mmol) and DMAP (3 mg, 0.025 mmol) in methylenechloride (0.8 mL), a solution of methyldithioproprionic acid (3.6 mg,0.024 mmol) in methylene chloride (4.0 mL) was added. The reaction wasstirred under N2 at room temperature for 5 hours. The reaction wasquenched with saturated aqueous ammonium chloride and extracted intomethylene chloride (25 mL×2). The combined organic layers were washedwith water (15 mL×1), dried over anhydrous magnesium sulfate andconcentrated in vacuo. The residue was purified on a silica gel columnwith 30% ethyl acetate in hexanes as the eluant yielding product 12′(21.3 mg, 75%): 1H NMR (CDCl3) δ1.12 (s, 27 H), 1.23 (s, 6 H), 1.37 (s,9 H), 1.68 (s, 3 H), 1.72 (s, 3 H), 1.88 (s, 3 H), 1.93 (s, 3 H), 2.17(s, 3 H), 2.41 (s, 3 H), 2.69 (t, J=6.8 Hz, 2 H), 2.86 (m, 2 H) 3.22 (brs, 1 H), 3.80 (s, 3 H), 3.95 (m, 4 H), 4.31 (d, J=8.0 Hz, 1 H), 4.38 (d,J=2.4 Hz, 1 H), 4.45 (d, J=8.4 Hz, 1 H), 4.76 (t, J=9.8 Hz, 1 H), 4.90(m, 2 H), 5.29 (s, 1 H), 5.34 (d, J=9.2 Hz, 1 H), 5.48 (dd, J=7.2, 10.8Hz, 1 H), 5.66 (d, J=6.4 Hz, 1 H), 6.11 (t, J=8.8 Hz, 1 H), 6.95 (d,J=9.2 Hz, 1 H), 7.06 (dd, J=3.2, 9.2 Hz, 1 H), 7.28 (d, J=3.2 Hz, 1 H).m/z LC/MS for C56H85NO17S2SiNa+: cacld: 1158.50; found: 1158.33.

[0193] Taxane 3′(3′-dephenyl-3′-(isobutenyl)-2-debenzoyl-2-(2,5-dimethoxybenzoyl)-7-(methyldisulfonyl-propanoyl)-docetaxel)was prepared by the following steps. Under N2, compound 12′ (27.6 mg,0.0243 mmol) was dissolved in pyridine-acetonitrile (1/1, 2.0 mL).HF/pyridine (70:30, 0.28 mL) was added at 0 oC. and the reaction stirredfor 24 hours, warming to room temperature. The reaction was quenchedwith saturated aqueous sodium bicarbonate and extracted into ethylacetate (30 mL×3). The combined organic layers were washed withadditional saturated aqueous sodium bicarbonate (25 mL×1), followed bysaturated aqueous cupric sulfate (25 mL×3). The combined organic layerswere washed with water (25 mL×1), dried over anhydrous magnesium sulfateand concentrated in vacuo. The crude residue was purified on a silicagel column with 50% ethyl acetate in hexanes as the eluant, yielding3′(19.7 mg, 82.8 %): 1H NMR (CDCl3) δ1.25 (s, 6 H), 1.38 (s, 9 H), 1.69,(s, 3 H), 1.74 (s, 3 H), 1.87 (s, 3 H), 1.94 (s, 3 H), 2.18 δ (s, 3 H),2.41 (s, 3 H), 2.68 (t, J=6.8 Hz, 2 H), 2.86 (m, 2 H), 3.12 (br s, 1 H),3.29 (d, J=6.4 Hz, 1 H), 3.80 (s, 3 H), 3.92 (m, 4 H), 4.16 (d, J=2.0,6.4 Hz, 1 H), 4.30 (d, J=8.0 Hz, 1 H), 4.43 (d, J=8.4 Hz, 1 H), 4.75 (m,2 H), 4.90 (d, J=8.0 Hz, 1 H), 5.29 (s, 1 H), 5.33 (d, J=8.0 Hz, 1 H),5.46 (dd, J=7.2, 10.8 Hz, 1 H), 5.65 (d, J=6.4 Hz, 1 H), 6.14 (t, J=8.8Hz 1 H), 6.95 (t, J=9.2 Hz, 1 H), 7.06 (dd, J=3.2, 9.2 Hz, 1 H), 7.28(d, J=3.2 Hz, 1 H). m/z LC/MS for C47H65NO17S2Na+: calcd: 1002.37; found1001.99.

Example 3

[0194] In Vitro Cytotoxicity Assays

[0195] The sulfide, disulfide, and sulfhydryl containing taxane drugs ofthe invention can be evaluated for their ability to suppressproliferation of various human tumor cell lines in vitro. Four adherentcell lines, A431 (human epidermoid carcinoma), SKBR3 (human breasttumor), A549 (human lung carcinoma) and MCF-7 (human breast tumor), andthe non-adherent cell line, Namalwa (Burkitt's lymphoma) are used forthe assessment of cytotoxicity of these compounds. Cells are exposed tothe compounds for 72 hours and the surviving fractions of cells aremeasured in direct assays. A431, SKBR3, A549 and MCF-7 are assayed forplating efficiency (Goldmacher et al, J. Cell. Biol. 102: 1312-1319(1986) and Namalwa are assayed by growth back extrapolation (Goldmacheret al, J. Immunol. 135: 3648-3651 (1985). IC50 values are thencalculated from this data.

[0196] The cytotoxicity of taxanes 2′ and 3′ was measured as follows.

[0197] A431, A549 and MCF-7 cells were plated at different densities in6-well tissue-culture plates in DMEM medium supplemented with 10% fetalcalf serum. Taxane 2′, at varying concentrations, was added and thecells were maintained in a humidified atmosphere at 37 oC. and 6% CO2until colonies of approximately 20 cells or more were formed (6 to 10days). Control plates contained no taxane. The cells were then fixedwith formaldehyde, stained with crystal violet, and counted under alow-magnification microscope. Plating efficiencies were then determinedfrom the colony numbers and surviving fractions of cells were calculatedas the ratio of the plating efficiency of the treated sample and theplating efficiency of the control.

[0198]FIG. 10 shows the results of the cytotoxicity determination.Taxane 2′ bearing a free hydroxy group at C-10 and a linking group atC-7 is highly potent with an IC50 value of 8×10-10 M towards A431 cells.In contrast, the corresponding taxane 1′ (FIG. 3) that bears an estergroup at C-10 is non-toxic to these cells even at 3×10-9 M. Theseresults demonstrate that the C-10 position of a taxane does not need tobe protected to maintain high potency.

[0199] The cytotoxic potency of taxane 3′ was similarly confirmed. A549and MCF-7 cells were plated at different densities in 6-welltissue-culture plates in DMEM medium supplemented with 10% fetal calfserum. Taxane 2′ at varying concentrations, was added and the cells weremaintained in a humidified atmosphere at 37 oC. and 6% CO2 untilcolonies of approximately 20 cells or more were formed (6 to 10 days).Control plates contained no taxane. The cells were then fixed withformaldehyde, stained with crystal violet, and counted under alow-magnification microscope. Plating efficiencies were then determinedfrom the colony numbers and surviving fractions of cells were calculatedas the ratio of the plating efficiency of the treated sample and theplating efficiency of the control.

[0200]FIG. 11 shows the results of the cytotoxicity determination.Taxane 3′ bearing a free hydroxy group at C-10 and a linking group atC-7 shows even greater potency towards the two tumor cell lines testedwith IC50 values of 1.8×10-10 M and 6.3×1011 M [10-11 M?] towards A549and MCF-7 cells respectively. These results confirm that the C-10position of a taxane does not need to be protected to maintain highpotency.

Example 4

[0201] Conjugation to Antibodies

[0202] Conjugation of Thiol-Containing Taxane to Antibodies viaDisulfide Links

[0203] The conjugation of thiol-containing taxanes to antibodies, orfragments thereof, via disulfide links is performed in two steps. In thefirst step dithiopyridyl groups are introduced into antibodies orantibody fragments using succinimidyl pyridyldithiopentanoate (SPP) asdescribed by Carlsson et al. The thiopyridyl groups are then displacedby reaction with the thiol-containing taxane to produce a conjugate.

[0204] Preparation of Antibody-SS-Taxane Conjugates

[0205] Antibodies anti-B4, anti-EGF receptor and N901, or fragmentsthereof, are modified with SPDP or SPP as described in the literature.Between 1 to 10 dithiopyridyl groups are introduced on the average perantibody molecule.

[0206] A solution of the dithiopyridyl modified antibody at aconcentration of 1 mg/ml in 0.1 M potassium phosphate buffer pH 6.5containing 1 mM EDTA at 25 oC. is treated with a thiol-containing taxane(1.25 molar equivalent/dithiopyridyl group). The release of thiopyridinefrom the modified antibody or fragment thereof is monitoredspectrophotometrically at 343 nm and is found to be complete in about 20hours. The antibody-taxane conjugate is purified and freed of unreacteddrug and other low molecular weight material by gel filtration through acolumn of Sephadex G-25. The number of taxane molecules bound perantibody molecule is determined by measuring the ratio between theabsorbances at 230 nm and 275 nm. An average of 1-10 taxane moleculesper antibody molecule can be linked via disulfide bonds by this method.

[0207] Conjugation of Thiol-Containing Taxane to Antibodies via aNoncleavable Thioether Link

[0208] The conjugation of a thiol-containing taxane is performed in twosteps. The antibody, or fragment thereof, is first reacted withsuccinimidyl maleimidomethylcyclohexane carboxylate (SMCC) to introducemaleimido groups. The modified antibody is then reacted with thethiol-containing taxane forming thioether links.

[0209] Preparation of Antibody-Taxane Conjugates (Non-Cleavable)

[0210] Antibodies, anti-B4, anti-EGF receptor and N901, or fragmentsthereof, are modified with SMCC as described in the literature.

[0211] The modified antibodies or antibody fragments are treated withthiol-containing taxane (1.25 molar equivalent/maleimido group). Themixtures are incubated overnight at 4 oC. The antibody-taxane conjugatesare purified as described above. Typically, an average of 1-10 taxanemolecules per antibody molecule are linked.

Example 5

[0212] Specific Preparation of Antibody-Taxane Conjugates

[0213] Murine monoclonal antibodies directed against the human EGFreceptor (EGFR) were developed. The EGF receptor is known to beover-expressed in several human squamous cell cancers, such as, head andneck, lung and breast. Four different antibodies, KS-61 (IgG2a), KS-77(IgGl), KS-78 (Ig2a), and KS-62 (IgG2a) were linked to taxanes viadisulfide bonds. The murine monoclonal antibody TA1, directed againstthe neu oncogene over-expressed in human breast and ovarian cancers, wasused for the preparation of TA1-taxane conjugates. The preparation ofthese particular conjugates is described below.

[0214] Preparation Of Anti-EGFR Antibody KS-61 -Taxane Conjugate

[0215] The anti-EGFR antibody KS-61 was first modified withN-succinimidyl-4-[2-pyridyldithio] pentanoate (SPP) to introducedithiopyridyl groups. The antibody (2.3 mg/mL) in 50 mM potassiumphosphate buffer, pH 6.5, containing NaCl (50 mM) and EDTA (2 mM), wastreated with SPP (11 molar equivalents in ethanol). The final ethanolconcentration was 1.4% (v/v). After 90 minutes at ambient temperature,lysine (50 mM) was added to help in the removal of any non-covalentlybound SPP. The reaction was allowed to proceed for two hours, and thenpurified by gel filtration through a Sephadex G25 column equilibrated inthe above buffer. Antibody-containing fractions were pooled and thedegree of modification was determined by treating a sample withdithiothreitol and measuring the change in absorbance at 343 nm (releaseof pyridine-2-thione with ε343=8,080 M−1 cm-1). Recovery of the antibodywas about 90%, with 5.0 pyridyldithio groups linked per antibodymolecule.

[0216] The modified antibody was diluted with 50 mM potassium phosphatebuffer, pH 6.5, containing NaCl (50 mM) and EDTA (2 mM) to a finalconcentration of 1.28 mg/mL. Taxane-SH (1.7 eq. per dithiopyridyl group)in ethanol (10% v/v in final reaction mixture) was then added to themodified antibody solution. The reaction proceeded at ambienttemperature under argon for 24 hours. The progress of the reaction wasmonitored spectrophotometrically at 343 nm for release ofpyridine-2-thione, caused by disulfide exchange between the taxane-SHand the dithiopyridyl groups on the antibody. The increase in absorbanceat 343 nm indicated that the taxane had linked to the antibody. Thereaction mixture was then loaded on to a Sephadex G25 SF gel filtrationcolumn equilibrated with phosphate-buffered saline (PBS, pH 6.5)containing 20% propylene glycol. The major peak comprised monomericKS-61-Taxane. The concentration of the conjugate was determined bymeasuring the absorbance at 280 nm. The conjugate was formulated withTween 80 (0.05%) and human serum albumin (HSA, 1 mg/mL).

[0217] Preparation Of Anti-EGFR Antibody KS-77-Taxane Conjugate

[0218] The anti-EGFR antibody KS-77 was modified with N-succinimidyl4-[2-pyridyldithio] pentanoate (SPP) to introduce dithiopyridyl groups.The antibody (5.0 mg/mL) in 50 mM potassium phosphate buffer, pH 6.5,was treated with SPP (11 molar equivalents in ethanol). The finalethanol concentration was 2 % (v/v). After 90 minutes at ambienttemperature, lysine (50 mM) was added to help in the removal of anynon-covalently bound SPP. The reaction mixture was allowed to incubatefor two hours, and then purified by gel filtration through a SephadexG25 column equilibrated in the above buffer. Antibody containingfractions were pooled and the degree of modification was determined bytreating a sample with dithiothreitol and measuring the change inabsorbance at 343 nm (release of 2-mercaptopyridine with ε343=8,080 M−1cm-1). Recovery of the antibody was about 90%, with 4.24 pyridyldithiogroups linked per antibody molecule.

[0219] The modified antibody was diluted with 50 mM potassium phosphatebuffer, pH 6.5, containing NaCl (50 mM) and EDTA (2 mM) to a finalconcentration of 1.4 mg/mL. Taxane-SH (1.7 equivalents per dithiopyridylgroup) in ethanol (10% v/v in final reaction mixture) was then added tothe modified antibody solution. The reaction proceeded at ambienttemperature under argon for 24 hours. An increase in absorbance at 343nm was noted, indicating that pyridine-2-thione was being released, andthe taxane had linked to the antibody. The reaction mixture was thenloaded on to a Sephacryl S300HR gel filtration column equilibrated withphosphate-buffered saline (PBS, pH 6.5). The major peak comprisedmonomeric KS-77-Taxane. The concentration of antibody KS-77 wasdetermined by measuring the absorbance at 280 nm. The conjugate wasformulated with Tween 80 (0.06%) and HSA (1 mg/mL).

[0220] Preparation Of Anti-EGFR Antibody KS-62-Taxane Conjugate

[0221] The anti-EGF antibody-taxane conjugate (KS-62-Taxane) wasprepared in a manner similar to that described for the preparation ofthe anti-EGFR antibody KS-77-taxane conjugate above. The modifiedantibody was diluted with 50 mM potassium phosphate buffer, pH 6.5,containing NaCl (50 mM) and EDTA (2 mM) to a final concentration of 2.5mg/mL. The antibody was modified with SPP to introduce 5.25pyridyldithio groups per antibody molecule Taxane-SH (1.7 eq.) inethanol (10% v/v in final reaction mixture) was then added to themodified antibody solution. The reaction proceeded at ambienttemperature under argon for 24 hours. The conjugate was purified bypassage through a Sephacryl S300HR gel filtration column equilibratedwith phosphate-buffered saline (PBS, pH 6.5). The major peak comprisedmonomeric KS-62-Taxane. The conjugate was formulated in PBS, containingTween 80 (0.01%, w/v) and HSA (1 mg/mL).

[0222] Preparation of Anti-EGFR Antibody KS-78-Taxane Conjugate

[0223] The anti-EGFR antibody-Taxane conjugate, KS-78-Taxane, wasprepared in a manner similar to that described for the preparation ofthe anti-EGFR antibody KS-77-taxane conjugate above. The modifiedantibody was diluted with 50 mM potassium phosphate buffer, pH 6.5,containing NaCl (50 mM) and EDTA (2 mM) to a final concentration of 1.6mg/mL. The antibody was modified with SPP to introduce 4.0 pyridyldithiogroups per antibody molecule. Taxane-SH (1.7 eq.) in ethanol (15% v/v infinal reaction mixture) was then added to the modified antibodysolution. The reaction proceeded at ambient temperature under argon for24 hours. The solution was then split into two batches, Batch A andBatch B, which were treated separately. Batch A was dialyzed againstPBS, pH 6.5 containing 2 mM CHAPS (3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate) and 20% (v/v) propylene glycol. ThepH of the final solution was 6.0. Batch B was dialyzed into PBS, pH 6.5containing 20% (v/v) propylene glycol. After dialyses, HSA (1 mg/mL) wasadded to both batches. Batch B was further treated with Tween 80 (0.05%,w/v).

[0224] Preparation of TA1-Taxane Conjugate

[0225] The murine monoclonal antibody TA1, which binds to the neuoncogene expressed on breast and ovarian tumors, was used in thepreparation of a taxane conjugate. TA1 (3.2 mg/mL) in 50 mM potassiumphosphate buffer, pH 6.5, containing NaCl (50 mM) and EDTA (2 mM) wastreated with SPP (8.0 molar equivalents in ethanol). The final ethanolconcentration was 5% (v/v). After 90 minutes at ambient temperature,lysine (50 mM) was added to help in the removal of any non-covalentlybound SPP. The reaction mixture was incubated for 2 hours, and then gelfiltered through a Sephadex G25 column equilibrated in the above buffer.Antibody-containing fractions were pooled and the degree of modificationwas determined by treating a sample with dithiothreitol and measuringthe change in absorbance at 343 nm (release of pyridine-2-thione withε343=8,080 M−1 cm-1). Recovery of the antibody was about 90%, with 4.9pyridyldithio groups linked per antibody molecule.

[0226] The modified antibody was diluted with 50 mM potassium phosphatebuffer, pH 6.5, containing NaCl (50 mM) and EDTA (2 mM) to a finalconcentration of 1.0 mg/mL. Taxane-SH (1.7 eq. per pyridyldithio groupincorporated) in ethanol (10% v/v in final reaction mixture) was thenadded to the modified antibody solution. The reaction proceeded atambient temperature under argon for 24 hours. The release ofpyridine-2-thione (monitored at 343 nm), indicated that the disulfideexchange between the Taxane-SH and the pyridyldithio substituent on theantibody was complete. A portion of the reaction mixture (4.0 mg) wasthen loaded on a Sephacryl S300HR gel filtration column equilibratedwith phosphate-buffered saline (PBS, pH 6.5). The major peak comprisedmonomeric TA1-Taxane. The remaining conjugate was diluted to 0.5 mg/mL,and dialyzed into 50 mM potassium phosphate buffer, pH 6.5, containingNaCl (50 mM), EDTA (2 mM) and 20% propylene glycol. The concentration ofantibody TA1 was determined in both species by measuring the absorbanceat 280 nm. The conjugates were formulated in PBS containing Tween 80(0.01%) and HSA (1 mg/mL).

Example 6

[0227] Other Methods of Linking Taxanes

[0228] Acid Labile Linkers

[0229] Taxanes can be esterified with N-protected amino acids, such asN-tboc-L-alanine in the presence of dicyclohexyl-carbodiimide anddimethylaminopyridine (DMAP) by standard methods described in thechemical literature. Cleavage of the t-boc protecting group withtrifluoroacetic acid will give a taxane ester containing a terminalamino group. This amino group containing taxane can be linked toantibodies, or fragments thereof, and other cell binding agents via anacid labile linker as previously described (Blättler et al,Biochemistry, 24: 1517-1524 (1985), U.S. Pat. Nos. 4,542,225, 4,569,789and 4,764,368).

[0230] Photolabile Linker

[0231] The amino group-containing taxane derivative described above canbe linked to cell binding agents via a photolabile linker as previouslydescribed. (Senter et al, Photochemistry and Photobiology, 42: 231-237(1985), U.S. Pat. No. 4,625,014).

[0232] Peptidase Labile Linker

[0233] The amino group-containing taxane described above can also belinked to cell binding agents via peptide spacer linkers. It has beenpreviously shown that short peptide spacers between drugs andmacromolecular protein carriers are stable in serum but are readilyhydrolyzed by intracellular lysosomal peptidases (Trouet et al, Proc.Nat'l. Acad. Sci., 79: 626-629 (1982)). The amino group containingtaxane can be condensed with peptides such as Ala-Leu, Leu-Ala-Leu or adimer of Ala-Leu using condensing agents such as1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide-HCl to give a peptidederivative of the taxane which can then be linked to cell bindingagents.

[0234] Esterase Labile Linker

[0235] Taxanes can be esterified by reaction of the hydroxyl group withsuccinic anhydride and then linked to a cell binding agent to produce aconjugate that can be cleaved by intracellular esterases to liberatefree drug. (For examples see: Aboud-Pirak et al, Biochem. Pharmacol.,38: 641-648 (1989), Laguzza et al, J. Med. Chem., 32: 549-555 (1989)).

Example 7

[0236] In Vivo Anti-Tumor Activity

[0237] The anti-tumor effect of anti-EGF receptor antibody-taxaneconjugate on human squamous cancer (A431) xenografts in SCID mice wasestablished as follows. The anti-tumor effect of two differentanti-human epidermal growth factor receptor-taxane conjugates(anti-EGFR-taxane conjugates), KS-61-Taxane and KS-77-Taxane wasevaluated in a human tumor xenograft model in SCID mice.

[0238] Five week old female SCID mice (25 animals) were inoculatedsubcutaneously in the right flank with A-431 human squamous cancer cells(1.5×106 cells/mouse) in 0.1 mL of serum-free medium. The tumors weregrown for 11 days to an average size of 100.0 mm3 (range of 54-145 mm3).The animals were then randomly divided into four groups (3 to 5 animalsper group) according to their tumor size. The first group receivedKS-61-Taxane conjugate (10 mg/kg, qd×5) administered intravenously. Thesecond group received the KS-77-Taxane conjugate (10 mg/kg, qd×5)administered intravenously. The third group received free(non-conjugated) taxane (0.24 mg/kg, qd×5, intravenously) at the samedose as that present in the conjugate. The fourth group, a control groupof animals, received PBS using the same treatment schedule as groups1-3.

[0239] The sizes of the tumors were measured twice weekly and the tumorvolumes were calculated with the formula: ½(length×width×height). Theweight of the animals was also measured twice per week. The results areshown in FIGS. 12 and 13. The tumors in the control group of mice grewto a size of nearly 1000 mm3 in 31 days. Treatment with free taxaneshowed no therapeutic effect, and the tumors in this group grew atessentially the same rate as in the control group of animals thatreceived PBS.

[0240] In contrast, both of the anti-EGFR-taxane conjugates showedremarkable anti-tumor activity resulting in complete inhibition of tumorgrowth in all the treated animals for the duration of the experiment—34days for the KS-61-Taxane conjugate and 27 days for the KS-77-Taxaneconjugate. The data also show that targeted delivery of the taxane usinga tumor-specific antibody is essential for the anti-tumor activity,since an equivalent dose of unconjugated taxane showed no anti-tumoreffect in this model. Importantly, the doses of antibody-taxaneconjugate used were non-toxic to the animals as demonstrated by theabsence of any weight loss (see FIG. 13).

Example 8

[0241] In Vitro Cytotoxicity of Antibody-Taxane Conjugates

[0242] The cytotoxicity of anti-EGFR-taxane conjugate, KS-78-Taxane, wasmeasured in a clonogenic assay using the EGF-receptor-positive humanA431 cell line (ATCC CRL 1555). N901-taxane conjugate, a similarconjugate made with the mouse monoclonal antibody N901 against humanCD56 was tested as a specificity control, since A431 cells do notexpress its target antigen, CD56. The cytotoxicity of TA1-Taxaneconjugate, a conjugate made with the mouse monoclonal antibody TA1against human Neu antigen, was measured on the antigen-positive humancell line SK-BR-3 (ATCC HTB 30) and the antigen-negative A431 cell line.Cells were plated at different densities in 6-well tissue-culture platesin DMEM medium supplemented with 10% fetal calf serum. Immunoconjugatesat varying concentrations were added and the cells were maintained in ahumidified atmosphere at 37 oC. and 6% CO2 until colonies ofapproximately 20 cells or more were formed (6 to 10 days). Controlplates contained no immunoconjugate. The cells were then fixed withformaldehyde, stained with crystal violet, and counted under alow-magnification microscope. Plating efficiencies were then determinedfrom the colony numbers and surviving fractions of cells were calculatedas the ratio of the plating efficiency of the treated sample and theplating efficiency of the control.

[0243]FIG. 14 shows the results of the cytotoxicity determination forthe two Batches of KS-78-Taxane conjugate on the target antigen-positivecell line A431. Conjugates from both batches show similar toxicity tothe target cells; treatment for 6 days at concentrations of 10-8 Machieved surviving fractions of less than 10-2 (less than 1% of cellssurvive). A control conjugate, N901-Taxane, for which there are noantigens present on the surface of A431 cells, shows no toxicity to thecells at concentrations of up to 3×10-8 M. Unconjugated KS-78 antibodyalso shows very little cytotoxic effect. These results demonstrate thetarget antigen-specific cytotoxicity of the KS-78-taxane conjugate.

[0244] The cytotoxic potency and selectivity of the TA1-taxane conjugatewas assayed with the target antigen-positive cell line SK-BR-3 and thetarget antigen-negative cell line A431. The results are shown in FIG.15. At a conjugate concentration of 10-9 M, more than 90% of the targetSK-BR-3 cells were killed (surviving fraction of less than 0.1), whileno toxicity towards the non-target A431 cells was observed. Theseresults demonstrate the selective killing of antigen-positive cells andthat the cytotoxic effect of the conjugate is dependent on the specificbinding through its antibody component.

Examples 9 and 10

[0245] General Methods:

[0246] Chemicals were obtained from Aldrich Chemical Co. or othercommercial sources and were used without further purification, unlessotherwise noted. All anhydrous reactions were performed in oven-driedglassware under argon. Tetrahydrofuran (THF) was distilled oversodium/benzophenone. All reactions were monitored by E. Merck analyticalthin layer chromatography (TLC) plates (silica gel 60 GF, aluminum back)and analyzed with 254 nm UV light and/or vanillin/sulfuric acid sprayand/or phosphomolybdic acid/ethanol spray. Silica gel for columnchromatography was purchased from E. Merck (230-400 mesh). Preparativethin layer chromatography (PTLC) plates (silica gel 60 GF) werepurchased from Analtech. 1H and 13C NMR spectra were obtained in CDCl3on a Bruker 400 MHz spectrometer and were assigned by comparison ofchemical shifts and coupling constants with those of related compounds.Chemical shifts are reported as δ-values, and coupling constants arereported in Hertz. Mass spectra were obtained on an Agilent Esquire 3000Electrospray Mass Spectrometer. The phrase “worked-up in the usual way”refers to diluting the reaction mixture with an excess amount of anorganic solvent, washing with water and brine, drying over sodiumsulfate and evaporating the solvent in vacuo unless otherwise noted. Thebeta-lactams 4, 19 and 38 the baccatin III derivative 7 were preparedfollowing the procedures that are reported in the literature (Brieva, R.Crich, J. Z.; Sih, C. J. J. Org. Chem., 58: 1068-1075 (1993); Holton, R.A.; Zhang, Z.; Clarke, P. A.; Nadizadeh, H.; Procter, J. D. TetrahedronLett. 39: 2883-2886 (1998); Chen, S -H.; Vittorio, F.; Wei, J -M.; Long,B.; Fairchild, C.; Mamber, S. W.; Kadow, J. F.; Vyas, D.; Doyle, T. W.Bioorganic Med. Chem. Lett., 4(3): 479-482 (1994). NMR data of these.compounds were identical to those in the literature.

Example 9

[0247] Synthesis of New Taxoids 12-15, 31-35 and 50-54 (FIGS. 5 and 16)of the Present Invention is Described Below.

[0248] General procedure for the coupling of the baccatin III derivative7 with the β-lactams 6a-d 21-25 and 40-44.

[0249] Synthesis of the silyl protected taxoids 8-11 26-30 and 45-49. Toa stirred solution of the baccatin derivative 7 (0.04 mmol) in THF (2mL) was added NaH (2 mmol). The reaction mixture was stirred for 15min., a β-lactam, such as 6a-d, 21-25 or 40-44; 0.08 mmol) wasintroduced, and the reaction mixture was further stirred for 4-6 h. Thereaction was diluted with EtOAc, quenched with acetic acid, andworked-up in the usual way. Finally the crude product was applied on aPTLC plate (30% EtOAc/Hexane) and the desired product was isolated.

[0250] General Procedure for Removal of the Silyl Protecting Groups;Synthesis of the Taxoids 12-15, 31-35 and 50-54

[0251] To a stirred solution of every 10 mg of a protected taxoid 8-11,26-30 or 45-49) in THF (0.5 mL) was added 0.15 mL of pyridine at 0 oC.Then over 5 min, 0.15 mL of HF-pyridine was introduced to the stirredsolution. The reaction mixture was allowed to come to room temperatureand further stirred for 24 h. The reaction mixture was then diluted withEtOAc, washed with saturated aqueous NaHCO3 solution and worked-up inthe usual way. Finally the crude product was applied to a PTLC plate(60% EtOAc/Hexane) and the desired product was isolated.

[0252] Synthesis of Representative Disulfide-Containing Taxoids (FIGS.6, 17) of the Present Invention.

[0253] Removal of the C-10 Acetate Group. Synthesis of 16.

[0254] To a stirred solution of the taxoid 10 (˜70 mg) in ethanol (1.5mL) was added at room temperature hydrazine monohydrate (0.6 mL). Thereaction mixture was stirred at room temperature for 1 h, then dilutedwith ethyl acetate, washed with aqueous saturated ammonium chloridesolution and worked-up in the usual way. The crude product was appliedto a PTLC plate (10% EtOAc/CH2Cl2) and the desired product was isolated.

[0255] Esterification of the C-10 Hydroxyl Group of Taxoids. Synthesisof 17 and 36.

[0256] To a stirred solution of a carboxylic acid in dichloromethane (2mL for every 30 mg of acid) was added EDC(1-[3-(dimethylamino)propyl-3-ethylcarbodiimide hydrochloride) (1equiv.) at room temperature and the reaction mixture was stirred for 15min. DMAP (4-(dimethylamino)pyridine) (catalytic amount) was then addedand the reaction mixture was stirred for another 5 min. The C-10deacetyl taxoid 16 (1/15 equiv.) was then introduced at room temperatureand the reaction mixture was further stirred for 4 h. The reactionmixture was diluted with ethyl acetate, washed with water, saturatedaqueous NaHCO3 solution and worked-up in the usual way. Finally thecrude product was applied to a PTLC plate (10% EtOAc/Hexane) and thedesired product was isolated.

[0257] Synthesis of Disulfide-containing Taxoids 18 and 37

[0258] To a stirred solution of every 10 mg of a protected taxoid 17 or36 in THF (0.5 mL) was added 0.15 mL of pyridine at 0 oC. Then over 5min, 0.15 mL of HF-pyridine was introduced to the stirred solution. Thereaction mixture was allowed to come to room temperature and furtherstirred for 24 h. The reaction mixture was then diluted with EtOAc,washed with saturated aqueous NaHCO3 solution and worked-up in the usualway. Finally the crude product was applied to a PTLC plate (60%EtOAc/Hexane) and the desired products 18 and 37 were obtained.

[0259] Compound 6a.

[0260] 1H NMR (CDCl3) δ7.03 (m, 1 H), 5.26 (dt, 1H), 4.96 (t, 1H), 4.94(t, 1H), 1.82 (s, 3H), 1.76 (s, 3H), 1.63 (m, 8 H), 1.06 (m, 21 H)

[0261] Compound 6b.

[0262] 1H NMR (CDCl3) δ7.31 (m, 1 H), 5.26 (dt, 1H), 4.96 (t, 1H), 4.92(t, 1H), 2.6 (m, 6H), 1.82 (s, 3H), 1.76 (s, 3H), 1.06 (m, 21 H)

[0263] Compound 6c.

[0264] 1H NMR (CDCl3) δ7.1 (m, 1H), 6.74 (dd, 1H), 5.24 (dt, 1H), 5.02(d, J=6 Hz 1H), 4.85 (m, 1H), 1.91 (dd, 3H), 1.81 (s, 3H), 1.77 (s, 3H),1.06 (m, 21 H); 13C NMR (CDCl3) δ166.93, 162.99, 145.98, 140.20, 124.01,117.68, 76.89, 55.77, 26.05, 18.33, 18.28, 17.66, 17.50, 17.46; LRMS m/zcalculated for C20H35NO3SiNa (M+Na)+388.23, found 388.

[0265] Compound 6d.

[0266] 1H NMR (CDC13) δ6.55 (m, 1H), 5.24 (dt, 1H), 4.98 (d, J=5.6 Hz,1H), 4.85 (m, 1H), 2.17 (s, 3H), 1.94 (s, 3H), 1.81 (s, 3H), 1.77 (s,3H) 1.06 (m, 21 H); 13C NMR (CDC13) δ166.65, 163.29, 159.81, 139.58,126.15, 118.27, 117.45,76.59, 55.71, 27.92, 26.07, 21.25, 18.34, 17.50,17.46; LRMS m/z calculated for C21H37NO3SiNa (M+Na)+402.24, found 402.1.

[0267] Compound 8.

[0268] 1H NMR (CDCl3) δ7.32 (d, J=3.2 Hz, 1H), 7.07 (d, J=3.2 Hz, 1H),7.04 (d, J=3.2 Hz, 1H), 6.95 (d, J=9.2 Hz, 1H), 6.58 (bs, 1H), 6.43 (s,1H), 6.12 (d, J=9.2 Hz, 1H), 6.03 (t, 1H), 5.67 (d, J=6.4 Hz, 1H), 5.38(d, J=8.8 Hz, 1H), 5.09 (t, 1H), 4,89 (d, J=8.4 Hz, 1H), 4.47 (s, 1H),4.42 (m, 2H), 4.28 (d, J=8 Hz, 1H), 3.97 (s, 3H), 3.79 (s, 3H), 3.74 (m,1H), 2.48 (m, 1H), 2.36 (d, 1H), 2.20 (s, 3H), 2.17 (s, 6H), 2.08 (m,2H), 1.98 (s, 3H), 1.89 (m, 2H), 1.72 (s, 9H), 1.60 (m, 5H), 1.22 (s,3H), 1.21 (s, 3H), 1.11 (s, 21H), 0.91 (t, 9H), 0.56 (m, 6H).

[0269] Compound 9.

[0270] 1H NMR (CDCl3) δ7.32 (d, J=3.2 Hz, 1H), 7.07 (d, J=2.8 Hz, 1H),7.04 (d, J=3.2 Hz, 1H), 6.95 (d, J=8.8 Hz, 1H), 6.47 (bs, 1H), 6.43 (s,1H), 6.07 (d, J=9.2 Hz, 1H), 6.4 (t, 1H), 5.68 (d, J=6.8 Hz, 1H), 5.38(d, J=9.2 Hz, 1H), 5.09 (t, 1H), 4.89 (d, J=8.4 Hz, 1H), 4.47 (s, 1H),4.42 (m, 2H), 4.28 (d, J=8.4 Hz, 1H), 3.96 (s, 3H), 3.79 (s, 3H), 3.74(d, J=6.4 Hz, 1H), 2.45 (m, 5H), 2.35 (d, J=9.2 Hz, 1H), 2.17 (s, 3H),2.15 (s, 3H), 1.98 (s, 3H), 1.89 (m, 4H), 1.72 (s, 9H), 1.24 (s, 3H),1.22 (s, 3H), 1.11 (s, 21H), 0.90 (t, 9H), 0.55 (m, 6H).

[0271] Compound 10.

[0272] 1H NMR (CDC13) δ7.29 (d, J=3.2 Hz, 1H), 7.05 (dd, 1H), 6.94 (d,J=9.2 Hz, 1H), 6.71 (m, 1H), 6.43 (s, 1H), 6.05 (t, 1H), 5.74 (m, 2H),5.67 (d, J=6.8 Hz, 1H), 5.37 (d, J=8.8 Hz, 1H), 5.10 (t, 1H), 4.88 (d,J=9.6 Hz, 1H), 4.25-4.47 (m, 5H), 4.10 (m, 1H), 3.96 (s, 3H), 3.79 (s,3H), 3.73 (m, 1H), 2.48 (m, 1H), 2.36 (bs, 1H), 2.33 (bs, 1H), 2.17 (s,3H), 2.15 (s, 3H), 1.97 (s, 3H), 1.79 (d, J=6.8 Hz, 3H), 1.71 (s, 9H),1.21 (m, 7H), 1.10 (s, 21H), 0.90 (t, 12H), 0.55 (m, 6H); 13C NMR(CDCl3) δ201.97, 171.67, 169.68, 169.27, 166.77, 164.65, 153.40, 152.83,140.51, 139.82, 136.79, 133.72, 124.99, 121.57, 120.26, 119.93, 115.91,113.54, 84.35, 81.00, 77.22, 76.44, 76.24, 75.27, 74.72, 72.19, 71.91,58.62, 56.78, 55.81, 50.16, 46.54, 42.86, 37.30, 36.47, 26.55, 25.59,22.54, 21.13, 20.82, 18.47, 18.01, 17.93, 17.63, 14.37, 12.53, 9.98,6.68, 5.27; LRMS m/z calculated for C59H91NO16Si2Na (M +Na)+1148.58,found 1148.5.

[0273] Compound 11.

[0274] 1H NMR (CDCl3) δ7.29 (d, J=3.2 Hz, 1H), 7.05 (dd, 1H), 6.94 (d,J=9.2 Hz, 1H), 6.44 (s, 1H), 6.04 (t, 1H), 5.65 (m, 2H), 5.49 (s, 1H),5.38 (d, J=9.2 Hz, 1H), 5.13 (t, 1H), 4.89 (d, J=8.4 Hz, 1H), 4.45 (s,1H), 4.40 (m, 2H), 4.27 (d, J=8 Hz, 1H), 3.98 (s, 3H), 3.79 (s, 3H),3.74 (m, 1H), 3.14 (s, 1H), 2.48 (m, 3H), 2.17 (s, 3H), 2.15 (s, 3H),2.04 (s, 3H), 1.98 (s, 3H), 1.71 (s, 9H), 1.21 (s, 3H), 1.10 (s, 3H),1.10 (s, 21H), 0.90 (t, 9H), 0.55 (m, 6H); 13C NMR (CDCl3) δ203.94,202.03, 171.80, 169.66, 169.28, 166.86, 165.62, 153.42. 152.75, 151.23,150.56, 140.71, 136.39, 136.17, 133.64, 132.41, 121.83, 120.35, 119.96,118.46, 115.84, 113.58, 113.49, 106.05, 84.37, 81.03, 77.18, 76.45,76.40, 75.32, 74.90, 72.20, 72.06, 60.88, 58.64, 56.74, 55.82, 49.78,46.55, 45.82, 42.84, 37.32, 36.51, 26.96, 26.47, 25.59, 22.55, 21.12,20.83, 19.68, 18.02, 17.85, 14.40, 12.54, 9.98, 6.69, 5.28; LRMS m/zcalculated for C6OH93NO16Si2Na (M+Na)+1162.59, found 1162.3.

[0275] Compound 12.

[0276] 1H NMR (CDCl3) δ7.32 (d, J=3.2 Hz, 1H), 7.07 (dd, 1H), 6.94 (d,J=9.2 Hz, 1H), 6.58 (s, 1H), 6.29 (s, 1H), 6.18 (t, 1H), 5.89 (d,J=8.4Hz, 1H), 5.66 (d, J=6.8 Hz, 1H), 5.38 (d, 1H), 5.10 (t, 1H), 4.93(d, 1H), 4.40 (d, J=8.4 Hz, 1H), 4.35 (m, 1H), 4.27 (d, J=8.4 Hz, 1H),4.24 (m, 1H), 3.94 (s, 3H), 3.80 (s, 3H), 3.74 (d, J=6.8 Hz, 1H), 3.61(d, J=6.4 Hz, 1H), 3.00 (s, 1H), 2.58−2.30 (m, 4H), 2.23 (s, 3H), 2.20(s, 3H), 2.13 (m, 4 H), 1.86 (s, 3H), 1.75 (s, 3H), 1.72 (s, 3H), 1.69(s, 3H), 1.63 (s, 6H), 1.60 (m, 2H), 1.29 (s, 3H), 1.15 (s, 3H); LRMSm/z calculated for C47H61NO16Na (M+Na)+918.39, found 918.3.

[0277] Compound 13.

[0278] 1H NMR (CDCl3) δ7.33 (d, J=3.2 Hz, 1H), 7.06 (dd, 1H), 6.94 (d,J=9.2 Hz, 1H), 6.50 (s, 1H), 6.29 (s, 1H), 6.19 (t, 1H), 5.86 (d, J=8.4Hz, 1H), 5.66 (d, J=6.8 Hz, 1H), 5.38 (d, J=9.2 Hz, 1H), 5.08 (t, 1H),4.93 (d, J=8.4 Hz, 1H), 4.39 (m, 2H), 4.29 (d, J=8.4 Hz, 1H), 4.25 (m,1H), 3.93 (s, 3H), 3.80 (s, 3H), 3.74 (d, J=6.4 Hz, 1H), 3.64 (d, J=6.4Hz, 1H), 3.00 (s, 1H), 2.41-2.56 (m, 7H), 2.32 (m, 1H), 2.23 (s, 3H),2.22 (s, 3H), 1.98 (m, 2H), 1.86 (s, 3H), 1.75 (m, 1H), 1.29 (s, 3H),1.15 (s, 3H); 13C NMR (CDCl3) δ203.90, 172.88, 171.24, 170.21, 166.63,164.86, 153.39, 153.03, 142.25, 139.02, 138.93, 138.60, 133.33, 120.24,120.14, 119.90, 115.93, 113.55, 84.51, 81.04, 77.74, 76.47, 76.10,75.77, 73.56, 72.23, 72.17, 58.83, 56.69, 55.86, 49.95, 45.56, 42.88,36.56, 35.74, 33.14, 31.48, 26.90, 25.67, 23.25, 22.42, 21.72, 20.86,18.55, 14.97, 9.53; LRMS m/z calculated for C46H59NO16Na (M+Na)+904.37,found 904.4.

[0279] Compound 14.

[0280] 1H NMR (CDCl3) δ7.33 (d, J=3.2 Hz, 1H), 7.07 (dd, 1H), 6.95 (d,J=9.2 Hz, 1H), 6.78 (m, 1H), 6.29 (s, 1H), 6.19 (t, 1H), 5.74 (d, J=15.2Hz, 1H), 5.67 (d, J=6.4 Hz, 1H), 5.62 (d, J=8.4 Hz, 1H), 5.39 (d, J=8.8Hz, 1H), 5.09 (t, 1H), 4.93 (d, 1H), 4.40 (d, J=8.4 Hz, 1H), 4.37 (m,1H), 4.30 (d, J=8.4 Hz, 1H), 4.24 (m, 1H), 3.94 (s, 3H), 3.80 (s, 3H),3.74 (d, J=6.4 Hz, 1H), 3.59 (d, J=6 Hz, 1H), 2.97 (s, 1H), 2.31-2.56(m, 5H), 2.23 (s, 3H), 2.21 (s, 3H), 1.84 (s, 3H), 1.82 (m, 3H), 1.75(s, 3H), 1.72 (s, 3H), 1.69 (s, 3H), 1.60 (s, 3H), 1.29 (s, 3H), 1.15(s, 3H); LRMS m/z calculated for C44H57NO16Na (M+Na)+878.36, found878.3.

[0281] Compound 15.

[0282] 1H NMR (CDC13) δ7.33 (d, J=3.2 Hz, 1H), 7.07 (dd, 1H), 6.94 (d,J=8.8 Hz, 1H), 6.29 (s, 1H), 6.18 (t, 1H), 5.67 (d, J=6.8Hz, 1H), 5.52(m, 2H), 5.40 (d, J=8.8 Hz, 1H), 5.05 (t, 1H), 4.93 (d, 1H), 4.40 (m,2H), 4.29 (d, J=8.4 Hz, 1H), 4.23 (m, 1H), 3.94 (s, 3H), 3.80 (s, 3H),3.75 (d, J=6.4 Hz, 1H), 3.57 (d, J=6.4 Hz, 1H), 2.97 (s, 1H), 2.33-2.58(m, 4H), 2.23 (s, 3H), 2.21 (s, 3H), 2.07 (s, 3H), 1.87 (s, 3H), 1.81(s, 3H), 1.75 (s, 3H), 1.72 (s, 3H), 1.68 (s, 3H), 1.40 (t, 1H), 1.29(s, 3H), 1.15 (s, 3H); 13C NMR (CDCl3) δ203.95, 196.43, 173.15, 171.25,170.08, 166.71, 166.28, 153.41, 152.97, 152.13, 151.25, 142.50, 138.44,136.19, 133.19, 120.45, 120.19, 119.96, 117.80, 115.85, 113.53, 106.08,94.84, 91.01, 84.52, 81.01, 77.71, 76.45, 76.24, 75.79, 73.61, 72.50,72.18, 58.82, 56.64, 55.86, 49.85, 45.84, 45.55, 42.86, 36.63, 35.71,27.12, 26.80, 25.60, 22.41, 21.75, 20.85, 19.73, 18.48, 14.95, 9.52;LRMS m/z calculated for C45H59NO16Na (M+Na)+892.37, found 892.3.

[0283] Compound 16.

[0284] 1H NMR (CDC13) δ7.30 (t, 1H), 7.06 (dm, 1H), 6.95 (d, J=9.2 Hz,1H), 6.72 (m, 1H), 6.12 (q, 1H), 5.75 (m, 2H), 5.64 (d, J=6.8 Hz, 1H),5.62 (d, J=8.4 Hz, 1H), 5.37 (m, 1H), 5.10 (m, 2H), 4.89 (d, 1H),4.26-4.47 (m, 5H), 3.97 (s, 3H), 3.80 (s, 3H), 3.79 (d, 1H), 3.10 (d,J=15.6 Hz, 1H), 2.31-2.44 (m, 3H), 2.18 (d, 3H), 2.09 (t, 1H), 1.91 (s,3H), 1.69-1.82 (m, 12H), 1.57 (m, 1H), 1.25 (s, 3H), 1.12 (m, 25H), 0.9(m, 12H), 0.5 (m, 6H).

[0285] Compound 17.

[0286] 1H NMR (CDCl3) δ7.30 (t, 1H), 7.06 (dm, 1H), 6.95 (d, J=9.2 Hz,1H), 6.72 (m, 1H), 6.47 (s, 1H), 6.05 (t, 1H), 5.72 (m, 2H), 5.38 (m,1H), 5.10 (m, 1H), 4.89 (d, 1H), 4.40-4.48 (m, 3H), 4.28 (d, J=8 Hz,1H), 3.98 (s, 3H), 3.80 (s, 3H), 3.74 (d, J=6.4 Hz, 1H), 3.13 (d, J=12Hz, 1H), 2.82-3.15 (m, 4H), 2.43 (s, 3H), 2.35-2.51 (m, 3H), 2.18 (d,3H), 2.09 (m, 1H), 1.99 (s, 3H), 1.79-1.93 (m, 2H), 1.71 (m, 9H), 1.56(s, 3H), 1.22 (s, 6H), 1.11 (s, 21H), 0.9 (m, 12H), 0.5 (m, 6H).

[0287] Compound 18.

[0288] 1H NMR (CDCl3) δ7.32 (t, 1H), 7.06 (dm, 1H), 6.95 (d, J=8.8 Hz,1H), 6.72 (m, 1H), 6.33 (s, 1H), 6.05 (q, 1H), 5.67 (d, J=6.8 Hz, 1H),5.60 (d, 1H), 5.39 (m, 1H), 5.07 (m, 1H), 4.93 (d, 1H), 4.20-4.42 (m,4H), 3.95 (s, 3H), 3.80 (s, 3H), 3.74 (d, J=6.4 Hz, 1H), 2.91-3.04 (m,5H), 2.42 (s, 3H), 2.31-2.53 (m, 3H), 2.18 (d, 3H), 2.10 (m, 1H), 1.87(s, 3H), 1.79-1.93 (m, 2H), 1.75 (s, 3H), 1.72 (s, 3H), 1.67 (d, 3H),1.51-1.64 (m, 3H), 1.29 (s, 3H), 1.15 (s, 3H), 0.9 (t, 2H); LRMS m/zcalculated for C46H61NO16S2Na (M+Na)+970.33, found 970.2.

[0289] Compound 19.

[0290] 1H NMR (CDC13) δ7.41 (s, 1H), 7.30 (m, 2H), 6.80 (m, 2H), 6.38(m, 2H), 5.24 (d, J=4.8 Hz, 1H), 5.20 (d, J=5.2 Hz, 1H), 3.75 (s, 3H),1.03 (s, 21H); 13C NMR (CDCl3) δ165.33, 156.24, 148.27, 142.83, 130.91,118.45, 114.29, 110.64, 110.26, 77.94, 57.06, 55.41, 17.55, 17.49,11.80; LRMS m/z calculated for C23H33NO4SiNa (M+Na)+438.21, found 438.1.

[0291] Compound 20.

[0292] 1H NMR (CDCl3) δ7.39 (s, 1H), 6.54 (bs, 1H), 6.35 (m, 2H), 5.15(m, 1H), 4.81 (d, J=4.4 Hz, 1H), 0.98 (s, 21H); 13C NMR (CDCl3) δ169.81,150.49, 142.53, 110.48, 109.09, 80.03, 53.52, 17.49, 17.43, 11.73; LRMSm/z calculated for C16H27NO3SiNa (M+Na)+332.17, found 332.0.

[0293] Compound 21.

[0294] 1H NMR (CDCl3) δ7.38 (s, 1H), 7.13 (m, 1H), 6.80 (dd, 1H), 6.35(m, 2H), 5.25 (d, J=5.6 Hz, 1H), 5.19 (d, J=5.6 Hz, 1H), 1.93 (dd, 3H),0.98 (m, 21H); 13C NMR (CDCl3) δ166.40, 162.72, 147.47, 146.94, 142.79,123.57, 110.43, 109.82, 77.51, 54.96, 18.35, 17.45, 17.38, 11.71; LRMSm/z calculated for C2OH3INO4SiNa (M+Na)+400.19, found 400.0.

[0295] Compound 22.

[0296] 1H NMR (CDCl3) δ7.38 (s, 1H), 6.61 (m, 1H), 6.34 (m, 2H), 5.23(d, J=5.6 Hz, 1H), 5.15 (d, J=6 Hz, 1H), 2.18 (s, 3H), 1.95 (s, 3H),0.98 (m, 21H); 13C NMR (CDCl3) δ166.08, 162.86, 160.95, 147.82, 142.67,120.73, 117.02, 115.12, 110.40, 109.62, 77.11, 54.80, 27.96, 27.53,21.33, 17.44, 17.37, 11.70; LRMS m/z calculated for C21H33NO4SiNa(M+Na)+414.21 , found 414.0.

[0297] Compound 23.

[0298] 1H NMR (CDCl3) δ8.00 (m, 2H), 7.58 (tt, 1H), 7.42-7.48 (m, 3H),6.45 (d, J=3.2 Hz, 1H), 6.38 (m, 1H), 5.47 (d, J=6 Hz, 1H), 5.23 (d, J=6Hz, 1H), 0.99 (s, 21H); 13C NMR (CDCl3) δ166.22, 164.95, 147.77, 142.93,133.34, 131.96, 129.89, 128.13, 110.47, 110.00, 76.81, 55.17, 17.48,17.41, 11.73; LRMS m/z calculated for C23H3iNO4SiNa (M+Na)+436.19, found436.0.

[0299] Compound 24.

[0300] 1H NMR (CDCl3) δ7.40 (s, 1H), 6.36 (m, 2H), 5.14 (d, J=5.6 Hz,1H), 5.11 (d, J=5.6 Hz, 1H), 1.43 (s, 9H), 0.96 (m, 21H); 13C NMR(CDCl3) δ165.76, 147.97, 147.75, 142.73, 110.45, 109.72, 83.46, 77.83,56.16, 27.87, 17.44, 17.38, 11.69; LRMS m/z calculated for C21H35NO5SiNa(M+Na)+432.22, found 432.1.

[0301] Compound 25.

[0302] 1H NMR (CDCl3) δ8.03 (d, 1H), 7.65 (m, 1H), 7.39 (m, 1H), 6.56(m, 1H), 6.42 (d, J=3.2 Hz, 1H), 6.34 (m, 1H), 5.45 (d, J=5.6 Hz, 1H),5.23 (d, J=6 Hz, 1H), 0.99 (s, 21H); 13C NMR (CDC13) δ164.52, 154.39,147.56, 147.48, 145.45, 142.88, 120.86, 112.10, 110.44, 110.08, 76.56,17.45, 17.38, 11.71; LRMS m/z calculated for C21H29NO5SiNa(M+Na)+426.17, found 426.0.

[0303] Compound 26.

[0304] 1H NMR (CDCl3) δ7.32 (s, 1H), 7.04 (dd, 1H), 6.93 (d, J=9.2 Hz,1H), 6.78 (m, 1H), 6.45 (s, 1H), 6.31 (m, 1H), 6.16 (m, 2H), 5.86 (dd,1H), 5.68 (d, J=6.8 Hz, 1H), 5.56 (d, J=9.2 Hz, 1H), 5.00 (s, 1H), 4.89(d, J=8 Hz, 1H), 4.46 (m, 1H), 4.10 (d, J=8 Hz, 1H), 4.28 (d, J=8 Hz,1H), 3.94 (s, 3H), 3.79 (s, 3H), 3.76 (m, 1H), 3.47 (m, 1H), 2.48 (m,1H), 2.25 (s, 3H), 2.11 (s, 3H), 1.95 (s, 3H), 1.87 (m, 4H), 1.74 (s,3H), 1.22 (s, 6H), 1.10 (s, 21H), 1.03 (t, 12H), 0.55 (m, 6H).

[0305] Compound 27.

[0306] 1H NMR (CDCl3) δ7.32 (s, 1H), 7.04 (dd, 1H), 6.93 (d, J=9.2 Hz,1H), 6.45 (s, 1H), 6.31 (m, 1H), 6.16 (m, 2H), 6.05 (d, J=9.2 Hz, 1H),5.57-5.69 (m, 3H), 4.99 (s, 1H), 4.91 (d, J=8 Hz, 1H), 4.44 (m, 2H),4.28 (d, J=8 Hz, 1H), 3.94 (s, 3H), 3.79 (s, 3H), 3.76 (m, 1H), 2.31 (s,3H), 2.11 (s, 3H), 2.09 (s, 3H), 2.04 (s, 3H), 1.87 (s, 3H), 1.74 (s,3H), 1.22 (s, 6H), 1.10 (s, 21H), 1.03 (t, 12H), 0.55 (m, 6H); LRMS m/zcalculated for C60H89NO17Si2Na (M+Na)+1174.56, found 1174.3.

[0307] Compound 28.

[0308] 1H NMR (CDCl3) δ7.76 (d, 1H), 7.43-7.56 (m, 3H), 7.04 (dd, 1H),6.93 (dd, 1H), 6.44 (s, 1H), 6.22 (m, 1H), 5.69 (m, 1H), 4.90 (m, 1H),4.44 (m, 2H), 4.30 (d, J=8 Hz, 1H), 3.87 (s, 3H), 3.79 (s, 3H), 3.76 (m,1H), 2.31 (s, 1H), 2.19 (s, 3H), 2.02 (m, 2H), 1.74 (s, 3H), 0.88-1.13(m, 33H), 0.58 (m, 6H); LRMS m/z calculated for C62H87NO17Si2Na(M+Na)+1196.54, found 1196.3.

[0309] Compound 29.

[0310] 1H NMR (CDCl3) δ7.32 (s, 1H), 7.04 (dd, 1H), 6.92 (d, J=9.2 Hz,1H), 6.45 (s, 1H), 6.32 (m, 1H), 6.22 (s, 1H), 6.18 (t, 1H), 5.67 (d,J=6.4 Hz, 1H), 5.25 (q, 2H), 4.94 (s, 1H), 4.91 (d, J=8 Hz, 1H), 4.44(m, 2H), 4.29 (d, J=8 Hz, 1H), 3.93 (s, 3H), 3.78 (s, 3H), 3.76 (m, 1H),2.50 (m, 1H), 2.37 (m, 1H), 2.29 (s, 3H), 2.17 (s, 3H), 2.00 (s, 3H),1.74 (s, 3H), 1.41 (s, 9H), 1.38 (m, 2H), 1.22 (s, 3H), 1.20 (s, 3H),1.06 (m, 6H), 0.83-0.98 (m, 30H), 0.55 (m, 6H); LRMS m/z calculated forC6OH91NO18Si2Na (M+Na)+1192.57, found 1192.3.

[0311] Compound 30.

[0312] 1H NMR (CDC13) δ7.48 (s, 1H), 7.35 (s, 1H), 7.27 (d, J=3.2 Hz,1H), 7.14 (d, J=9.6 Hz), 7.06 (d, 1H), 7.04 (d, J=3.2 Hz, 1H), 6.94 (d,J=9.2 Hz, 1H), 6.51 (m, 1H), 6.45 (s, 1H), 6.33 (m, 1H), 6.24 (s, 1H),6.20 (t, 1H), 5.69 (d, J=6.4 Hz, 1H), 5,64 (d, J=9.2 Hz), 5.06 (s, 1H),H), 4.91 (d, 1H), 4.44 (m, 2H), 4.29 (d, J=8 Hz, 1H), 3.95 (s, 3H), 3.81(s, 3H), 3.79 (m, 1H), 3.18 (s. 1H), 2.50 (m, 1H), 2.37 (m, 1H), 2.33(s, 3H), 2.16 (s, 3H), 2.01 (s, 3H), 1.92 (m, 1H), 1.74 (s, 3H), 1.25(s, 3H), 1.23 (s, 3H), 0.88-1.02 (m, 27H), 0.55 (m, 6H); LRMS m/zcalculated for C6OH85NO18Si2Na (M+Na)+1186.52, found 1186.3.

[0313] Compound 31.

[0314] 1H NMR (CDCl3) δ7.39 (s, 1H), 7.30 (d, J=3.2 Hz, 1H), 7.07 (dd,1H), 6.95 (d, J=9.2 Hz, 1H), 6.84 (m, 1H), 6.35 (m, 1H), 6.32 (m, 1H),6.29 (s, 1H), 6.23 (t, 1H), 6.05 (d, J=9.2 Hz, 1H), 5.83 (dd, 1H), 5.65(m, 2H), 4.92 (d, 1H), 4.71 (s, 1H), 4.40 (m, 2H), 4.30 (d, J=8 Hz, 1H),3.91 (s, 3H), 3.75 (s, 3H), 3.73 (d, 1H), 3.40 (m, 1H), 3.06 (s, 1H),2.56 (m, 1H), 2.34 (m, 3H), 2.22 (s, 3H), 2.17 (s, 3H), 1.85 (s, 6H),1.70 (s, 3H), 1.29 (s, 3H), 1.25 (s, 1H), 1.16 (s, 3H); LRMS m/zcalculated for C44H53NO17Na (M+Na)+890.32, found 890.2.

[0315] Compound 32.

[0316] 1H NMR (CDCl3) δ7.39 (s, 1H), 7.30 (d, J=3.2 Hz, 1H), 7.07 (dd,1H), 6.95 (d, J=9.2 Hz, 1H), 6.36 (m, 1H), 6.31 (m, 2H), 6.22 (t, 1H),5.92 (d, J=9.2 Hz, 1H), 5.59-5.67 (m, 3H), 4.92 (d, 1H), 4.71 (m, 1H),4.40 (m, 2H), 4.30 (d, J=8 Hz, 1H), 3.91 (s, 3H), 3.81 (s, 3H), 3.74 (d,1H), 3.35 (d, 1H), 3.10 (s, 1H), 2.95 (s, 1H), 2.34-2.58 (m, 4H), 2.23(s, 3H), 2.20 (s, 3H), 2.09 (s, 3H), 1.86 (s, 3H), 1.85 (s, 3H), 1.73(s, 3H), 1.29 (s, 3H), 1.25 (s, 1H), 1.16 (s, 3H); LRMS m/z calculatedfor C45H55NO17Na (M+Na)+904.34, found 904.2.

[0317] Compound 33.

[0318] 1H NMR (CDCl3) δ7.75 (d, 1H), 7.43-7.56 (m, 4H), 7.29 (d, J=2.8Hz, 1H), 7.06 (dd, 1H), 6.95 (d, J=9.2 Hz, 1H), 6.38 (s, 1H), 6.29 (m,2H), 5.83 (d, 1H), 5.66 (d, 1H), 4.91 (d, 1H), 4.79 (m, 1H), 4.41 (d,J=8 Hz, 1H), 4.40 (m, 1H), 4.33 (d, J=8 Hz, 1H), 3.95 (s, 3H), 3.81 (s,3H), 3.77 (m, 1H), 3.12 (s, 1H), 2.42 (m, 1H), 2.25 (s, 3H), 2.24 (s,3H), 1.86 (s, 3H), 1.73 (s, 3H), 1.29 (s, 3H), 1.25 (s, 3H); LRMS m/zcalculated for C47H53NO17Na (M+Na)+926.32, found 926.2.

[0319] Compound 34.

[0320] 1H NMR (CDCl3) 6 7.39 (s, 1H), 7.28 (d, J=2.8 Hz, 1H), 7.07 (dd,1H), 6.95 (d, J=9.2 Hz, 1H), 6.36 (m, 1H), 6.31 (m, 2H), 6.21 (t, 1H),5.67 (d, J=6.4 Hz, 1H), 5.26 (d, 1H), 5.19 (d, 1H), 4.93 (d, 1H), 4.68(m, 1H), 4.40 (m, 2H), 4.30 (d, J=8 Hz, 1H), 3.93 (s, 3H), 3.80 (s, 3H),3.74 (d, 1H), 3.17 (m, 2H), 2.38-2.57 (m, 4H), 2.24 (s, 3H), 2.22 (s,3H), 1.88 (s, 3H), 1.73 (s, 3H), 1.41 (s, 9H), 1.29 (s, 3H), 1.25 (s,3H) 1.16 (s, 3H); LRMS m/z calculated for C45H57NO18Na (M+Na)+922.35,found 922.2.

[0321] Compound 35.

[0322] 1H NMR (CDCl3) δ7.47 (s, 1H), 7.42 (s, 1H), 7.30 (d, J=3.2 Hz,1H), 7.05-7.09 (m, 2H), 6.94-6.99 (m, 2H), 6.51 (m, 1H), 6.38 (m, 2H),6.28 (m, 2H), 5.75 (dd, 1H), 5.68 (d, J=6.4 Hz, 1H), 4.93 (d, 1H), 4.77(m, 1H), 4.40 (m, 2H), 4.31 (d, J=8.4 Hz, 1H), 3.95 (s, 3H), 3.81 (s,3H), 3.74 (d, 1H), 3.46 (d, 1H), 3.08 (s, 1H), 2.55 (m, 1H), 2.36-2.43(m, 3H), 2.23 (s, 6H), 1.85 (s, 3H), 1.73 (s, 3H), 1.29 (s, 3H), 1.16(s, 3H), 13C NMR (CDCl3) δ204.21, 172.66, 171.62, 170.70, 167.21,157.98, 153.84, 153.23, 151.08, 147.39, 144.88, 143.11, 142.26, 134.01,120.59, 120.40, 116.10, 115.69, 113.95, 112.73, 111.16, 108.33, 84.95,81.51, 78.01, 76.89, 76.61, 76.11, 73.14, 72.58, 72.03, 59.28, 57.11,56.28, 49.81, 45.99, 43.26, 36.97, 36.15, 27.36, 22.97, 22.10, 21.25,15.33, 9.94; LRMS m/z calculated for C45H51NO18Na (M+Na)+916.30, found916.2.

[0323] Compound 36.

[0324] 1H NMR (CDCl3) δ7.30 (t, 1H), 7.05 (dm, 1H), 6.95 (dd, 1H), 6.72(m, 1H), 6.45 (s, 1H), 6.05 (bt, 1H), 5.76 (d, 1H), 5.67 (d, J=6.4 Hz,1H), 5.38 (m, 1H), 5.09 (m, 1H), 4.88 (d, 1H), 4.39-4.48 (m, 3H), 4.27(d, J=8.4 Hz, 1H), 3.98 (s, 3H), 3.81 (s, 3H), 3.64-3.80 (m, 22H), 3.56(d, 1H), 2.90 (t, 2H), 2.74 (m, 2H), 2.41 (s, 3H), 2.16 (d, 3H), 2.10(m, 1H), 2.00 (s, 3H), 1.90 (m, 1H), 1.81 (m, 1H), 1.71 (s, 6H), 1.69(d, 3H), 1.61 (m, 2H), 1.21-1.31 (m, 14H), 1.21 (s, 21H), 0.92 (m, 16H),0.58 (m, 6H).

[0325] Compound 37.

[0326] 1H NMR (CDCl3) δ7.32 (t, 1H), 7.07 (dm, 1H), 6.95 (d, J=9.2 Hz,1H), 6.78 (m, 1H), 6.31 (s, 1H), 6.16 (q, 1H), 5.66 (d, 1H), 5.61 (d,1H), 5.39 (m, 1H), 5.06 (m, 1H), 4.93 (d, 1H), 4.39 (m, 2H), 4.25 (d,1H), 4.21 (ddd, 1H), 3.94 (s, 3H), 3.85 (d, 1H), 3.80 (s, 3H), 3.73 (t,3H), 3.64-3.65 (m, 13H), 2.98 (d, 1H), 2.90 (t, 2H), 2.80 (t, 2H), 2.39(s, 3H), 2.21 (d, 3H), 2.06 (m, 1H), 1.86 (s, 3H), 1.84 (m, 2H), 1.75(s, 3H), 1.71 (s, 3H), 1.69 (d, 3H), 1.61 (m, 2H), 1.27 (s, 3H), 1.14(s, 3H), 0.89 (t, 2H); 13C NMR (CDCl3) δ203.72, 173.09, 172.89, 172.28,171.68, 170.16, 170.04, 166.74, 166.69, 165.27, 153.42, 153.01, 152.94,142.36, 142.32, 140.84, 138.82, 138.63, 133.27, 124.49, 120.38, 120.26,120.19, 115.88, 115.85, 113.57, 84.52, 81.03, 77.74, 77.63, 76.46,75.79, 73.49, 73.28, 72.46, 72.24, 72.17, 70.64, 70.62, 70.56, 70.51,70.40, 69.76, 66.36, 58.82, 56.70, 56.66, 55.86, 50.08, 49.76, 45.58,42.84, 38.39, 37.59, 36.58, 35.76, 35.01, 26.93, 26.86, 25.64, 25.59,23.47, 22.43, 21.77, 19.14, 18.51, 18.46, 17.74, 14.97, 14.94, 13.58,9.54

[0327] Compound 38.

[0328] 1H NMR (CDCl3) δ7.31 (m, 3H), 7.10 (d, 1H), 6.99 (dd, 1H), 6.78(m, 2H), 5.41 (d, J=4.8 Hz, 1H), 5.23 (d, J=5.2 Hz, 1H), 3.73 (s, 3H),1.01 (s, 21H); 13C NMR (CDCl3) δ165.34, 156.22, 137.45, 130.81, 127.49,126.64, 126.15, 118.65, 114.26, 78.02, 59.28, 55.37, 17.55, 17.46,11.79; LRMS m/z calculated for C23H33NO3SSiNa (M+Na)+454.18, found454.0.

[0329] Compound 40.

[0330] 1H NMR (CDCl3) δ7.28 (dd, 1H), 7.11 (m, 2H), 6.98 (dd, 1H), 6.78(dd, 1H), 5.50 (d, J=5.6 Hz, 1H), 5.21 (d, J=6 Hz, 1H), 1.93 (dd, 3H),1.01 (s, 21H); 13C NMR (CDCl3) δ166.40, 162.70, 147.00, 136.39, 127.61,126.54, 125.88, 123.61, 77.39, 57.01, 18.35, 17.67, 17.47, 17.37, 12.26,11.72; LRMS m/z calculated for C20H31NO3SSiNa (M+Na)+416.17, found416.1.

[0331] Compound 41.

[0332] 1H NMR (CDCl3) δ7.28 (dd, 1H), 7.11 (m, 1H), 7.00 (dd, 1H), 6.80(s, 1H), 6.26 (dd, 1H), 5.87 (dd, 1H), 5.50 (d, J=6 Hz, 1H), 5.19 (d,J=5.6 Hz, 1H), 2.35 (s, 1H), 2.20 (dd, 6H), 0.98 (m, 21H); 13C NMR(CDCl3) δ166.04, 164.59, 162.83, 160.70, 148.34, 136.96, 127.39, 126.50,125.65, 122.38, 120.70, 117.18, 117.12, 115.25, 77.14, 56.90, 17.48,17.39, 12.34, 11.82; LRMS m/z calculated for C21H33NO3SSiNa(M+Na)+430.18, found 430.1.

[0333] Compound 42.

[0334] 1H NMR (CDCl3) δ7.99 (d, 2H), 7.57 (t, 1H), 7.47 (t, 2H), 7.30(dd, 2H), 7.18 (d, 1H), 7.01 (t, 1H), 5.73 (d, J=6 Hz, 1H), 5.25 (d,J=6.4 Hz, 1H), 1.04 (m, 21H); 13C NMR (CDCl3) δ166.13, 164.82, 136.86,129.89, 128.11, 127.80, 126.58, 125.96, 76.89, 57.11, 17.50, 17.41,11.83; LRMS m/z calculated for C23H31NO3SSiNa (M+Na)+452.17, found452.0.

[0335] Compound 43.

[0336] 1H NMR (CDC13) δ7.30 (dd, 1H), 7.08 (dd, 1H), 6.99 (dd, 1H), 5.34(d, J=5.6 Hz, 1H), 5.15 (d, J=5.6 Hz, 1H), 1.42 (s, 9H) 0.95 (m, 21H);13C NMR (CDCl3) δ165.75, 147.74, 136.80, 127.58, 126.48, 125.97, 122.05,83.53, 77.76, 58.26, 27.88, 17.67, 17.47, 17.37, 12.26,11.70; LRMS m/zcalculated for C21H35NO4SSiNa (M+Na)+448.20, found 448.1.

[0337] Compound 44.

[0338] 1H NMR (CDCl3) δ7.98 (d, 1H), 7.65 (dd, 1H), 7.29 (dd, 1H), 7.15(dd, 1H), 6.99 (dd, 1H), 6.56 (m, 1H), 5.71 (d, J=6 Hz, 1H), 5.25 (d,J=6 Hz, 1H), 0.99 (m, 21H); 13C NMR (CDCl3) δ164.11, 147.61, 127.96,126.57, 126.06, 120.95, 112.12, 76.52, 57.17, 17.67, 17.50, 17.39,11.74; LRMS m/z calculated for C21H29NO4SSiNa (M+Na)+442.15, found442.0.

[0339] Compound 45.

[0340] 1H NMR (CDCl3) δ7.27 (d, 1H), 7.20 (d, 1H), 7.05 (dd, 1H),6.87-6.95 (m, 4H), 6.76 (m, 1H), 6.45 (s, 1H), 6.27 (d, J=9.2 Hz, 1H),6.17 (t, 1H), 5.85 (d, 1H), 5.75 (d, J=9.2 Hz, 1H), 5.68 (d, J=6.8 Hz,1H), 4.92 (d, 1H), 4.83 (s, 1H), 4.40-4.45 (m, 2H), 4.28 (d, J=8 Hz,1H), 3.96 (s, 3H), 3.84 (d, 1H), 3.82 (s, 3H), 3.79 (d, 1H), 3.46 (m,1H), 3.19 (s, 1H), 2.34-2.49 (m, 3H), 2.30 (s, 3H), 2.17 (s, 3H), 2.00(s, 3H), 1.85-1.97 (m, 9H), 1.72 (s, 3H), 1.55-1.67 (m, 5H), 1.23 (s,6H), 1.00 (s, 21H), 0.92 (t, 9H), 0.58 (m, 6H); LRMS m/z calculated forC59H87NO16SSi2Na (M+Na)+1176.52, found 1176.4.

[0341] Compound 46.

[0342] 1H NMR (CDCl3) δ7.27 (d, 1H), 7.20 (d, 1H), 7.05 (dd, 1H),6.87-6.95 (m, 4H), 6.45 (s, 1H), 6.15-6.19 (m, 2H), 5.78 (d, 1H), 5.68(d, J=6.8 Hz, 1H), 5.60 (s, 1H), 4.90 (d, 1H), 4.82 (s, 1H), 4.40-4.46(m, 2H), 4.29 (d, J=8 Hz, 1H), 3.96 (s, 3H), 3.84 (d, 1H), 3.82 (s, 3H),3.76 (d, 1H), 3.46 (m, 1H), 3.23 (s, 1H), 2.49 (m, 1H), 2.34 (m, 2H),2.31 (s, 3H), 2.17 (m, 2H), 2.14 (s, 3H), 2.07 (s, 3H), 2.01 (s, 3H),1.85-1.97 (m, 9H), 1.83 (s, 3H), 1.71 (s, 3H), 1.55-1.67 (m, 3H), 1.39(m, 1H), 1.23 (s, 6H), 1.00 (s, 21H), 0.92 (t, 9H), 0.58 (m, 6H); LRMSm/z calculated for C6OH89NO16SSi2Na (M+Na)+1190.53, found 1190.5.

[0343] Compound 47.

[0344] 1H NMR (CDCl3) δ7.53-7.60 (m, 3H), 7.42-7.46 (t, 2H), 7.20 (d,1H), 7.13-7.15 (m, 3H), 6.92-7.01 (m, 3H), 6.54 (d, J=9.2 Hz, 1H), 6.45(s, 1H), 6.20 (d, 1H), 5.84 (m, 1H), 5.56 (d, J=6.4 Hz, 1H), 4.92 (m,1H), 4.84 (s, 1H), 4.65 (d, 1H), 4.64 (d, 1H), 4.22 (d, J=8 Hz, 1H),4.08 (m, 1H), 3.79 (m, 2H), 3.74 (s, 3H), 3.30 (m, 1H), 3.25 (s, 3H),2.67 (m, 1H), 2.46 (m, 2H), 2.18 (s, 3H), 2.17 (m, 2H), 2.07 (s, 3H),1.69 (s, 3H), 1.21 (s, 3H), 1.18 (s, 3H), 1.17 (s, 3H), 1.13 (m, 21H),0.92 (t, 9H), 0.58 (m, 6H); LRMS m/z calculated for C62H87NO16SSi2Na(M+Na)+1212.52, found 1212.5.

[0345] Compound 48.

[0346] 1H NMR (CDC13) δ7.27 (d, 1H), 7.21 (d, 1H), 7.06 (dd, 1H),6.90-6.97 (m, 3H), 6.46 (s, 1H), 6.17 (t, 1H), 5.68 (d, J=6.8 Hz, 1H),5.43 (d, 1H), 5.42 (d, 1H), 4.90 (d, 1H), 4.76 (s, 1H), 4.41-4.46 (m,2H), 4.29 (d, J=8 Hz, 1H), 3.94 (s, 3H), 3.79 (s, 3H), 3.77 (d, 1H),3.24 (s, 1H), 2.52 (m, 1H), 2.41 (m, 2H), 2.30 (s, 3H), 2.01 (s, 3H),1.88 (s, 1H), 1.73 (s, 3H), 1.40 (s, 9H), 1.23 (s, 3H), 1.22 (s, 3H),1.00 (s, 21H), 0.92 (t, 9H), 0.58 (m, 6H); LRMS m/z calculated forC6OH91NO17SSi2Na (M+Na)+1208.54, found 1208.5.

[0347] Compound 49.

[0348] 1H NMR (CDCl13) δ7.47 (s, 1H), 7.22-7.29 (m, 3H), 7.06 (d, 1H),7.05 (d, 1H), 6.94-6.98 (m, 3H), 6.50 (m, 1H), 6.44 (s, 1H), 6.19 (t,1H), 5.84 (d, 1H), 5.69 (d, J=6.8 Hz, 1H), 4.90 (d, 1H), 4.89 (s, 1H),4.41-4.46 (m, 2H), 4.29 (d, J=8.4 Hz, 1H), 3.98 (s, 3H), 3.82 (s, 3H),3.77 (d, 1H), 3.16 (s, 1H), 2.52 (m, 1H), 2.36 (m, 1H), 2.31 (s, 3H),2.24 (m, 1H), 2.15 (s, 3H), 2.00 (s, 3H), 1.88 (s, 1H), 1.72 (s, 3H),1.17 (s, 6H), 1.00 (s, 21H), 0.92 (t, 9H), 0.58 (m, 6H); LRMS m/zcalculated for C60H85NO17SSi2Na (M+Na)+1202.50, found 1202.4.

[0349] Compound 50.

[0350] 1H NMR (CDCl3) δ7.32 (d, 1H), 7.27 (dd, 1H), 7.06-7.10 (m, 2H),6.95-7.01 (m, 2H), 6.80 (m, 1H), 6.28 (s, 1H), 6.23 (t, 1H), 6.10 (d,J=9.2 Hz, 1H), 5.77-5.84 (m, 2H), 5.66 (d, J=6.8 Hz, 1H), 4.92 (d, 1H),4.67 (s, 1H), 4.40 (d, J=8.8 Hz, 1H), 4.35 (m, 1H), 4.29 (d, J=8 Hz,1H), 3.96 (s, 3H), 3.82 (s, 3H), 3.73 (d, J=6.4 Hz, 1H), 3.54 (bs, 1H),3.06 (bs, 1H), 2.55 (m, 1H), 2.38 (m, 3H), 2.24 (s, 3H), 2.20 (s, 3H),1.84 (dd, 3H), 1.82 (s, 3H), 1.29 (s, 3H), 1.16 (s, 3H); 13C NMR (CDCl3)δ203.82, 172.30, 171.23, 170.22, 166.79, 164.91, 153.44, 152.91, 141.88,141.56, 140.94, 133.58, 127.06, 125.82, 125.59, 124.25, 120.32, 119.91,115.68, 113.59, 99.99, 84.51, 81.15, 77.60, 76.50, 76.12, 75.72, 73.06,72.76, 72.20, 58.88, 56.74, 55.90, 50.77, 45.58, 42.87, 36.56, 35.75,26.96, 22.68, 21.67, 20.86, 17.82, 14.98, 9.53; LRMS m/z calculated forC44H53NO16SNa (M+Na)+906.3, found 906.2

[0351] Compound 51.

[0352] 1H NMR (CDCl3) δ7.32 (d, 1H), 7.27 (dd, 1H), 7.06-7.09 (m, 2H),6.94-6.99 (m, 2H), 6.29 (s, 1H), 6.22 (t, 1H), 6.01 (d, J=9.2 Hz, 1H),5.79 (d, 1H), 5.66 (d, J=6.8 Hz, 1H), 5.56 (s, 1H), 4.92 (d, 1H), 4.65(s, 1H), 4.40 (d, J=8 Hz, 1H), 4.35 (m, 1H), 4.30 (d, J=8.4 Hz, 1H),3.95 (s, 3H), 3.82 (s, 3H), 3.74 (d, J=6.4 Hz, 1H), 3.52 (bs, 1H), 3.07(bs, 1H), 2.34-2.57 (m, 4H), 2.24 (s, 3H), 2.21 (s, 3H), 1.84 (s, 6H),1.72 (s, 3H), 1.29 (s, 3H), 1.15 (s, 3H); 13C NMR (CDCl3) δ203.82,172.51, 171.23, 170.14, 166.79, 165.83, 153.43, 153.19, 152.87, 142.01,141.21, 133.49, 127.05, 125.64, 125.45, 120.32, 119.90, 117.48, 115.62,113.55, 84.51, 81.11, 77.56, 76.18, 75.73, 73.17, 72.90, 72.18, 58.85,56.68, 55.89, 50.57, 45.57, 42.86, 35.73, 27.21, 26.85, 22.67, 21.68,20.86, 19.84, 14.96, 9.52; LRMS m/z calculated for C45H55NO16SNa(M+Na)+920.31, found 920.2.

[0353] Compound 52.

[0354] 1H NMR (CDCl3) δ7.66 (dd, 2H), 7.53 (tt, 1H), 7.43 (t, 2H), 7.33(d, 1H), 7.22 (dd, 1H), 6.97-7.06 (m, 3H), 6.76 (d, 1H), 6.27 (s, 1H),6.16 (d, 1H), 5.91 (d, 1H), 5.62 (d, J=6.4 Hz, 1H), 4.96 (bs, 1H), 4.89(dd, 1H), 4.74 ( s, 1H), 4.52 (d, J=7.6 Hz, 1H), 4.16 (d, J=7.6 Hz, 1H),4.00 (m, 1H), 3.80 (s, 3H), 3.63 (s, 3H), 3.34 (d, J=6.4 Hz, 1H), 2.87(bs, 1H), 2.40-2.61 (m, 4H), 2.24 (s, 3H), 1.99 (m, 1H), 1.77 (s, 3H),1.66 (s, 3H), 1.13 (s, 3H), 1.12 (s, 3H); LRMS m/z calculated forC47H53NO16SNa (M+Na)+942.30, found 942.2.

[0355] Compound 53.

[0356] 1H NMR (CDCl3) δ7.30 (d, 1H), 7.27 (dd, 1H), 7.06-7.09 (m, 2H),6.94-7.00 (m, 2H), 6.30 (s, 1H), 6.21 (t, 1H), 5.66 (d, J=6.8 Hz, 1H),5.42 (d, 1H), 5.28 (d, 1H), 4.91 (d, 1H), 4.59 (s, 1H), 4.41 (d, J=8 Hz,1H), 4.38 (m, 1H), 4.30 (d, J=8.4 Hz, 1H), 3.94 (s, 3H), 3.81 (s, 3H),3.74 (d, J=6.4 Hz, 1H), 3.48 (bs, 1H), 3.11 (bs, 1H), 2.34-2.57 (m, 4H),2.22 (s, 3H), 2.19 (s, 3H), 1.89 (m, 1H), 1.85 (s, 3H), 1.72 (s, 3H),1.39 (s, 9H), 1.29 (s, 3H), 1.15 (s, 3H); 13C NMR (CDCl3) δ203.83,172.63, 171.20, 170.00, 166.81, 154.88, 153.45, 152.74, 142.06,141.29,133.46, 127.05, 125.36, 120.23, 119.99, 115.61, 113.47, 84.51,81.11, 80.36, 77.51, 76.43, 75.72, 73.21, 73.00, 72.15, 58.84, 56.54,55.85, 45.58, 42.85, 36.47, 35.72, 28.12, 26.73, 22.63, 20.84, 14.95,9.51; LRMS m/z calculated for C45H57NO17SNa (M+Na)+938.32, found 938.2.

[0357] Compound 54.

[0358] 1H NMR (CDCl3) δ7.46 (s, 1H), 7.32 (d, 1H), 7.28 (dd, 1H), 7.16(d, 1H) 6.95-7.09 (m, 4H), 6.50 (m, 1H), 6.28 (s, 1H), 6.25 (t, 1H),5.93 (d, 1H), 5.67 (d, J=6.8 Hz, 1H), 4.92 (d, 1H), 4.73 (s, 1H), 4.41(d, J=8.4 Hz, 1H), 4.38 (m, 1H), 4.30 (d, J=8.4 Hz, 1H), 3.96 (s, 3H),3.80 (s, 3H), 3.74 (d, J=6.4 Hz, 1H), 3.63 (bs, 1H), 3.09 (bs, 1H),2.33-2.58 (m, 4H), 2.22 (s, 3H), 2.19 (s, 3H), 1.89 (m, 1H), 1.82 (s,3H), 1.73 (s, 3H), 1.28 (s, 3H), 1.15 (s, 3H); LRMS m/z calculated forC45H51NO17SNa (M+Na)+932.28, found 932.2.

Example 10

[0359] In Vitro Cytotoxicity Assays

[0360] The new taxoids and the disulfide containing taxane drugs of theinvention were evaluated for their ability to suppress proliferation ofhuman tumor cell lines in vitro. The human tumor cell lines A-549 (humanlung carcinoma) and MCF-7 (human breast tumor), are used for theassessment of cytotoxicity of these compounds. Cells are exposed to thecompounds for 72 hours and the surviving fractions of cells are measuredin direct assays. A549 and MCF-7 are assayed for plating efficiency(Goldmacher et al, J. Cell. Biol. 102: 1312-1319 (1986) and IC50 valuesare then calculated from this data.

[0361] The cytotoxicity of taxoids 14, 15, 31-35, 50-54 anddisulfide-containing taxoids 18 and 37 was measured as follows. A549 andMCF-7 cells were plated at different densities in 6-well tissue-cultureplates in DMEM medium supplemented with 10% fetal calf serum. Thetaxane, at varying concentrations, was added and the cells weremaintained in a humidified atmosphere at 37 oC. and 6% CO2 untilcolonies of approximately 20 cells or more were formed (6 to 10 days).Control plates contained no taxane. The cells were then fixed withformaldehyde, stained with crystal violet, and counted under alow-magnification microscope. Plating efficiencies were then determinedfrom the colony numbers and surviving fractions of cells were calculatedas the ratio of the plating efficiency of the treated sample and theplating efficiency of the control.

[0362]FIG. 10 shows the results of the cytotoxicity determination oftwelve new taxoids of the present invention. Except for taxane 52, whichbears a phenyl substituent at R4, all the other new taxoids wereextremely potent towards both A-549 and MCF-7 cell lines with IC50values in the 10-10 to 10-11 M range. Taxane 52 was less cytotoxic withan IC50 value of 3×10-9 M towards both cell lines that were tested.

[0363]FIG. 11 shows the cytotoxicity curves for representativedisulfide-containing taxoids of the present invention.Disulfide-containing taxoids 18 and 37 are both extremely potent towardboth A-549 and MCF-7 cells and display steep killing curves.

What is claimed is:
 1. A compound represented by formula (I):

wherein: R₁ is H, an electron withdrawing group, or an electron donatinggroup; R₁′ and R₁″ are the same or different, and are H, an electronwithdrawing group, or an electron donating group; R₂ is H; R₃ is alkylor alkenyl having from 1 to 10 carbon atoms, cycloalkyl or cycloalkenylhaving from 3 to 10 carbon atoms, aryl, or heterocycle; R₄ is alkyl oralkenyl having from 1 to 10 carbon atoms, cycloalkyl or cycloalkenylhaving from 3 to 10 carbon atoms, aryl, heterocycle, —OC(CH₃)₃ or acarbamate formed from any of said alkyl, alkenyl, cycloalkyl,cycloalkenyl having from 3 to 10 carbon atoms, aryl, or heterocycle R₅is a linking group; and R₆ is H, a heterocyclic or aryl ether, ester orcarbamate, or, a linear, branched, or cyclic alkyl or alkenyl ester orether having from from 1 to 10 carbon atoms or a carbamate of theformula —COX, wherein X is a nitrogen-containing heterocycle, or acarbamate of the formula —CONR₁₀R₁₁, wherein R₁₀ and R₁₁ are the same ordifferent and are H, linear, branched, or cyclic alkyl having from 1 to10 atoms or simple or substituted aryl having from 1 to 10 carbon atoms.2. The compound of claim 1, wherein R₃ is —CH═C(CH₃)₂.
 3. A compoundrepresented by formula (I):

wherein: R₂ is a linking group; R₁ is H, an electron withdrawing groupor an electron donating group; R₁′ and R₁″ are the same or different andare H, an electron withdrawing group, or an electron donating group; R₃and R₄ are the same or different and are alkyl or alkenyl having from 1to 10 carbon atoms, cycloalkyl or cycloalkenyl having from 3 to 10carbon atoms, aryl, or heterocycle and R₄ additionally is —OC(CH₃)₃ or acarbamate formed from any of said alkyl, alkenyl, cycloalkyl,cycloalkenyl having from 3 to 10 carbon atoms, aryl, or heterocycle; R₅and R₆ are the same or different and are H, a heterocyclic or arylether, ester or carbamate, or a linear, branched, or cyclic alkyl oralkenyl ester or ether having from from 1 to 10 carbon atoms or acarbamate of the formula —COX, wherein X is a nitrogen-containingheterocycle, or a carbamate of the formula —CONR₁₀R₁₁, wherein R₁₀ andR₁₁ are the same or different and are H, linear, branched, or cyclicalkyl having from 1 to 10 carbon atoms or simple or substituted arylhaving from 1 to 10 carbon atoms.
 4. The compound represented by Formula(I),

wherein: R₅ is a linking group; R₁ is H, an electron withdrawing groupor an electron donating group; R₁′ and R₁″ are the same or different andare H, an electron withdrawing group, or an electron donating group; R3and R4 are the same or different and are alkyl or alkenyl having from 1to 10 carbon atoms, cycloalkyl or cycloalkenyl having from 3 to 10carbon atoms, aryl, or heterocycle and R₄ additionally is —OC(CH₃)₃ or acarbamate formed from any of said alkyl, alkenyl, cycloalkyl,cycloalkenyl having from 3 to 10 carbon atoms, aryl, or heterocycle; R₂and R₆ are the same or different and are H, a heterocyclic or arylether, ester or carbamate, or, a linear, branched, or cyclic alkyl oralkenyl ester or ether having from from 1 to 10 carbon atoms or acarbamate of the formula —COX, wherein X is a nitrogen-containingheterocycle, or a carbamate of the formula —CONR₁₀R₁₁, wherein R₁₀ andR₁₁, are the same or different and are H, linear, branched, or cyclicalkyl having from 1 to 10 atoms or simple or substituted aryl havingfrom 1 to 10 carbon atoms.
 5. A compound represented by formula (I):

wherein: R₆ is a linking group; R₁ is H, an electron withdrawing groupor an electron donating group; R₁′ and R₁″ are the same or different andare H, an electron withdrawing group, or an electron donating group; R₃and R₄ are the same or different and are alkyl or alkenyl having from 1to 10 carbon atoms, cycloalkyl or cycloalkenyl having from 3 to 10carbon atoms, aryl, or heterocycle and R₄ additionally is —OC(CH₃)₃ or acarbamate formed from any of said alkyl, alkenyl, cycloalkyl,cycloalkenyl, having from 3 to 10 carbon atoms, aryl, or heterocycle; R₂and R₅ are the same or different and are H, a heterocyclic or arylether, ester or carbamate, or, a linear, branched, or cyclic alkyl oralkenyl ester or ether having from from 1 to 10 carbon atoms or acarbamate of the formula —COX, wherein X is a nitrogen-containingheterocycle such as piperidino, morpholino, piperazino,N-methylpiperazino, or a carbamate of the formula —CONR₁₀R₁₁, whereinR₁₀ and R₁₁ are the same or different and are H, linear, branched, orcyclic alkyl having from 1 to 10 atoms or simple or substituted arylhaving from 1 to 10 carbon atoms;
 6. A compound represented by formula(I):

wherein: R₃ is a linking group; R₁ is H, an electron withdrawing groupor an electron donating group; R₁′ and R₁ 41 are the same or differentand are H, an electron withdrawing group, or an electron donating group;R₄ is alkyl or alkenyl having from 1 to 10 carbon atoms, cycloalkyl orcycloalkenyl having from 3 to 10 carbon atoms, aryl, or heterocycle andR₄ additionally is —OC(CH₃)₃ or a carbamate formed from any of saidalkyl, alkenyl, cycloalkyl, cycloalkenyl, having from 3 to 10 carbonatoms, aryl, or heterocycle; R₂, R₅ and R₆ are the same or different andare H, a heterocyclic or aryl ether, ester or carbamate, or, a linear,branched, or cyclic alkyl or alkenyl ester or ether having from from 1to 10 carbon atoms or a carbamate of the formula —COX, wherein X is anitrogen-containing heterocycle such as piperidino, morpholino,piperazino, N-methylpiperazino, or a carbamate of the formula—CONR₁₀R₁₁, wherein R₁₀ and R₁₁ are the same or different and are H,linear, branched, or cyclic alkyl having from 1 to 10 atoms or simple orsubstituted aryl having from 1 to 10 carbon atoms.
 7. A compoundrepresented by formula (I):

wherein: R₄ is a linking group; R₁ is H, an electron withdrawing groupor an electron donating group; R₁′ and R₁″ are the same or different andare H, an electron withdrawing group, or an electron donating group; R₃is alkyl or alkenyl having from 1 to 10 carbon atoms, cycloalkyl orcycloalkenyl having from 3 to 10 carbon atoms, aryl, or heterocycle andR₄ additionally is —OC(CH₃)₃ or a carbamate formed from any of saidalkyl, alkenyl, cycloalkyl, cycloalkenyl, having from 3 to 10 carbonatoms, aryl, or heterocycle; R₂, R₅ and R₆ are the same or different andare H, a heterocyclic or aryl ether, ester or carbamate, or, a linear,branched, or cyclic alkyl or alkenyl ester or ether having from from 1to 10 carbon atoms or a carbamate of the formula —COX, wherein X is anitrogen-containing heterocycle such as piperidino, morpholino,piperazino, N-methylpiperazino, or a carbamate of the formula—CONR₁₀R₁₁, wherein R₁₀ and R₁₁ are the same or different and are H,linear, branched, or cyclic alkyl having from 1 to 10 atoms or simple orsubstituted aryl having from 1 to 10 carbon atoms;